Novel molecules of the card-related protein family and uses thereof

ABSTRACT

Novel CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, CARD-5, and CARD-6 polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, CARD-5, and CARD-6 proteins, and the invention further provides CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, CARD-5, and CARD-6 fusion proteins, antigenic peptides and anti-CARD-3, anti-CARD-4L and anti-CARD-4S, anti-CARD-4Y, anti-CARD-4Z, anti-CARD-5, and anti-CARD-6 antibodies. The invention also provides CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, CARD-5, and CARD-6 nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a CARD-3, CARD-4L, CARD-4S, CARD-4Y, CARD-4Z, CARD-5, and CARD-6 gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/340,620, filed Jun. 28, 1999, which is a continuation-in-part of U.S.application Ser. No. 09/245,281, filed Feb. 5, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/207,359, filed Dec.8, 1998, which is a continuation-in-part of U.S. application Ser. No.09/099,041, filed Jun. 17, 1998, which is a continuation-in-part of U.S.application Ser. No. 09/019,942, filed Feb. 6, 1998. The contents ofeach of these applications is incorporated herein by reference.

BACKGROUND OF THE INVENTION

In multicellular organisms, homeostasis is maintained by balancing therate of cell proliferation against the rate of cell death. Cellproliferation is influenced by numerous growth factors and theexpression of proto-oncogenes, which typically encourage progressionthrough the cell cycle. In contrast, numerous events, including theexpression of tumor suppressor genes, can lead to an arrest of cellularproliferation.

In differentiated cells, a particular type of cell death calledapoptosis occurs when an internal suicide program is activated. Thisprogram can be initiated by a variety of external signals as well assignals that are generated within the cell in response to, for example,genetic damage. For many years, the magnitude of apoptotic cell deathwas not appreciated because the dying cells are quickly eliminated byphagocytes, without an inflammatory response.

The mechanisms that mediate apoptosis have been intensively studied.These mechanisms involve the activation of endogenous proteases, loss ofmitochondrial function, and structural changes such as disruption of thecytoskeleton, cell shrinkage, membrane blebbing, and nuclearcondensation due to degradation of DNA. The various signals that triggerapoptosis are thought to bring about these events by converging on acommon cell death pathway that is regulated by the expression of genesthat are highly conserved from worms, such as C. elegans, to humans. Infact, invertebrate model systems have been invaluable tools inidentifying and characterizing the genes that control apoptosis. Throughthe study of invertebrates and more evolved animals. numerous genes thatare associated with cell death have been identified, but the way inWvhich their products interact to execute the apoptotic program ispoorly understood.

Caspases, a class of proteins central to the apoptotic program, areresponsible for the degradation of cellular proteins that leads to themorphological changes seen in cells undergoing apoptosis. Caspases(cysteinyl aspartate-specific proteinases) are cysteine proteases havingspecificity for aspartate at the substrate cleavage site. Generallycaspases are classified as either initiator caspases or effectorcaspases both of which are zymo gens that are activated by proteolysisthat generates an active species. An effector caspase is activated by aninitiator caspase which cleaves the effector caspase. Initiator caspasesare activated by an autoproteolytic mechanism that is often dependentupon oligomerization directed by association of the caspase with anadapter molecule.

Many caspases and proteins that interact with caspases possess domainsof about 60 amino acids called a caspase recruitment domain (CARD).Hofmann et al. (TIBS 22:155, 1997) and others have postulated thatcertain apoptotic proteins bind to each other via their CARDs and thatdifferent subtypes of CARDs may confer binding specificity, regulatingthe activity of various caspases, for example. The functionalsignificance of CARDs have been repeatedly demonstrated. For example,Duan et al. (Nature 385:86, 1997) showed that deleting the CARD at theN-terminus of RAIDD abolished the ability of RAIDD to bind to caspases.

Caspase-1 is an example of an initiator caspase. Caspase-1 was firstdiscovered as the protease responsible for the conversion of theinactive precursor of IL-1β to the mature proinflammatory cytokine(caspase-1 was originally termed interleukin-1β converting enzyme, ICE).Caspase-1 also processes the inactive precursor of the cytokine IL-18into an active form. Caspase-1 is synthesized as a single chain zymogenconsisting of an N-terminal CARD containing prodomain and a large (p20)and small (p10) catalytic domain. Caspase-1 is thought to oligomerizeupon the receipt of a proinflammatory signal and autoprocess to generatean active heterodimeric protease consisting of the p20 and p10 subunits.

RIP2 (CARDIAK/RICK) binds caspase-1 via an interaction between the CARDdomain of RIP2 and the CARD domain of caspase-1 . This interactionresults in the processing and activation of caspase-1. Thus, RIP2 isthought to be an. upstream activator adaptor of caspase-1. Conversely,the activation of caspase-1 and subsequent generation of IL-1β isregulated by a CARD domain-containing decoy molecule termed ICEBERG.This decoy attenuates inflammation by binding to the CARD domain ofcaspase-1 and inhibiting or displacing the upstream activator RIP2.ICEBERG is induced by proinflammatory stimuli and thus appears to bepart of a negative feedback loop that shuts off IL-1β generation andthus dampens the inflammatory response (Humke et al., Cell 103:99,2000).

In addition to its role in inflammation via IL-1β processing, caspase-1also appears to participate in cell death pathways. For example.overexpression of caspase-1 in Rat-1 fibroblasts induces apoptosis thatcan be suppressed by overexpression of aintiapoptotic genes such asBcl-2 (Miura et al., Cell 75:653, 1993).

Caspase-9 activation may precede the activation of all other celldeath-related caspases in the mitochondrial pathways of apoptosis (Sleeet al., J. Cell Biol. 144:281-292, 1999). Inactive procaspase-9 isactivated by interaction with a complex which includes Apaf-1, aCARD-containing protein, and other factors (Li et al., Cell 91:479,1997; Srinivasula et al., Mol. Cell 1:949-959, 1998). Recognition ofprocaspase-9 by Apaf-1 occurs primarily through the interaction of theCARD of Apaf-1 with the prodomain of caspase-9. The CARD of Apaf-1shares about 20% sequence identity with the prodomain of procaspase-9.The prodomain of caspase-9 is a member of the CARD family of apoptoticsignaling motifs (Hofmann and Bucher, Trends in Biochem. Sci.22:155-156, 1997). A similar domain is present in caspase activatingproteins CED-4 and RAIDD/CRADD as well as in initiator caspases CED-3and caspase-2/ICH-1 (Duan and Dixit, Nature 385:86-89, 1997; Ahmad etal., Cancer Res. 57:615-619, 1997; Alnemri et al., Cell 87:171, 1996).Apaf-1 can bind several other caspases, e.g., caspase-4 and caspase-8(Inohara et al., J. Biol. Chem. 273:12296-12300, 1998).

Nuclear factor-κB (NF-κB) is a transcription factor expressed in manycell types and which activates homologous or heterologous genes thathave κB sites in their promoters. Molecules that regulate NF-κBactivation play a critical role in both apoptosis and inflammation.Quiescent NF-κB resides in the cytoplasm as a heterodimer of proteinsreferred to as p50 and p65 and is complexed with the regulatory proteinIκB. NF-κB binding to IκB causes NF-κB to remain in the cytoplasm. Atleast two dozen stimuli that activate NF-κB are known (New EnglandJournal of Medicine 336:1066, 1997) and they include cytokines, proteinkinase C activators, oxidants, viruses. and immune system stimuli. NF-κBactivating stimuli activate specific IκB kinases that phosphorylate IκBleading to its degradation. Once liberated from IκB. NF-κB translocatesto the nucleus and activates genes with κB sites in their promoters. Theproinflammatory cytokines TNF-α and IL-1 induce NF-κB activation bybinding their cell-surface receptors and activating the NF-κB-inducingkinase, NIK, and NF-κB. NIK phosphorylates the IκB kinases α and β whichphosphorylate IκB. leading to its degradation.

NF-κB and the NF-κB pathway has been implicated in mediating chronicinflammation in inflammatory diseases such as asthma, ulcerativecolitis, rheumatoid arthritis (Epstein, New England Journal of Medicine336:1066, 1997) and inhibiting NF-κB or NF-κB pathways may be aneffective way of treating these diseases. NF-κB and the NF-κB pathwayhas also been implicated in atherosclerosis (Navab et al., AmericanJournal of Cardiology 76:18C, 1995), especially in mediating fattystreak formation, and inhibiting NF-κB or NF-κB pathways may be aneffective therapy for atherosclerosis. Among the genes activated byNF-κB are cIAP-1, cIAP-2, TRAF1, and TRAF2, all of which have been shownto protect cells from TNF-α induced cell death (Wang et al., Science281:1680-83, 1998). CLAP, a protein which includes a CARD, activates theApaf-1-caspase-9 pathway and activates NF-κB by acting upstream of NIKand IκB kinase (Srinivasula et al., supra).

Bcl-2 family proteins are important regulators of pathways involved inapoptosis and can act to inhibit or promote cell death. Expression ofcertain anti-apoptotic Bcl-2 family members is commonly altered incancerous cells, suppressing programmed cell death and extending tumorgrowth. Among the anti-apoptotic Bcl-2 family members thus faridentified are Boo, Bcl-2, Bcl-x_(L), Bcl-w, NR-13, A1, and Mcl-2.Pro-apoptotic Bcl-2 family members include Bax, Bak, Bad, Bik. Bid, Hrk,Bim, and Bok/Mtd. Significantly, the anti-apoptotic Bcl-2 family member.Bcl-x_(L), has been shown to interact with Apaf-1 and blockApaf-1-dependent caspase-9 activation (Hu et al., Proc. Nat'l. Acad.Sci. 95:4386-4391, 1998). Boo, another anti-apoptotic Bcl-2 familymember interacts with Apaf-1 and caspase-9. Bak and Bik, pro-apoptoticBcl-2 family members, can disrupt the association of Boo with Apaf-1(Song et al. EMBO J. 18:167-178, 1999). Boo is thought to be involved inthe control of ovarian atresia and sperm maturation. Diva, anothermember of the Bcl-2 family, inhibits binding of Bcl-x_(L) to Apf-1,preventing Bcl-x_(L) from binding to Apaf-1.

Neurotrophins (e.g., NGF), which are best know as neuronal survivalfactors, can mediate apoptosis via the p75 neurotrophin receptor(p75^(NTR)). It is thought that p75^(NTR) activation can lead to NF-κBactivation (Carter et al., Science 272:542-545, 1996). It has beenproposed that p75^(NTR)-mediated cell death acts to ensure rapid celldeath when a neuron is unable to obtain sufficient neurotropins. Thismechanism could, for example, cause the elimination of neurons thatreach an inappropriate target or that reach an appropriate target at aninappropriate time (Miller and Kaplan, Cell Death and Diff. 5:343-345,1998).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofgenes encoding CARD-3, CARD-4, CARD-5, and CARD-6. A full-length humanCARD-3 cDNA is presented. Several CARD-4 cDNAs are presented. Briefly,the CARD-4 gene can express a long transcript that encodes CARD-4L, ashort transcript that encodes partial CARD-4S, or two CARD-4 splicevariants (CARD-4Y and CARD-4Z). A full length cDNA sequence for themurine ortholog of CARD-4L is also presented. Full-length cDNAs encodingmurine and human CARD-5 are presented. In addition, full-length cDNAsencoding human and rat CARD-6 are presented.

CARD-3, CARD-4, CARD-5, and CARD-6 are intracellular proteins that arepredicted to be involved in regulating caspase activation. CARD-4 isfound to activate the NF-κB pathway and to enhance caspase 9-mediatedcell death. CARD-5 is found to activate the NF-κB pathway and to bind tothe CARD domains of caspase-1, CARD-7, and CARD-5 itself. In addition,proteins that bind to CARD-4 are presented including CARD-3 and hNUDC.

The CARD-3 cDNA described below (SEQ ID NO:1) has a 1620 open readingframe (nucleotides 214 to 1833 of SEQ ID NO:1; SEQ ID NO:3) whichencodes a 540 amino acid protein (SEQ ID NO:2). CARD-3 contains a kinasedomain which extends from amino acid 1 to amino acid 300 of SEQ ID NO:2;SEQ ID NO:4, followed by a linker domain at amino acid 301 to amino acid431 of SEQ ID NO:2; SEQ ID NO:5 and a CARD at amino acid 432 to aminoacid 540 of SEQ ID NO:2; SEQ ID NO:6.

At least four forms of CARD-4 exist in the cell, a long form, CARD-4L, ashort form, CARD-4S, and two splice variants, CARD-4Y and CARD-4Z. ThecDNA of CARD-4L described below (SEQ ID NO:7) has a 2859 nucleotide openreading frame (nucleotides 245-3103 of SEQ ID NO:7; SEQ ID NO:9) whichencodes a 953 amino acid protein (SEQ ID NO:8). CARD-4L proteinpossesses a CARD domain (amino acids 15-114; SEQ ID NO:10). Thenucleotide sequence of the full length cDNA corresponding to the murineortholog of human CARD-4L is presented (SEQ ID NO:42) as is thepredicted amino acid sequence of murine CARD-4L (SEQ ID NO:43). Acomparison between the predicted amino acid sequences of human CARD-4Land murine CARD-4L is also depicted in FIG. 17.

Human CARD-4L is also predicted to have a nucleotide binding domainwhich extends from about amino acid 198 to about amino acid 397 of SEQID NO:8; SEQ ID NO:11, a Walker Box “A”, which extends from about aminoacid 202 to about amino acid 209 of SEQ ID NO:8; SEQ ID NO:12, a WalkerBox “B”, which extends from about amino acid 280 to about amino acid284, of SEQ ID NO:8; SEQ ID NO:13, a kinase 1a (P-loop) subdomain, whichextends from about amino acid 127 to about amino acid 212 of SEQ IDNO:8: SEQ ID NO:46, a kinase 2 subdomain, which extends from about aminoacid 273 to about amino acid 288 of SEQ ID NO:8; SEQ ID NO:47, a kinase3a subdomain, which extends from about amino acid 327 to about aminoacid 338 of SEQ ID NO:8; SEQ ID NO:14, and ten Leucine-rich repeatswhich extend from about amino acid 674 to about amino acid 950 of SEQ IDNO:8. The first Leucine-rich repeat extends from about amino acid 674 toabout amino acid 701 of SEQ ID NO:8; SEQ ID NO:15. The secondLeucine-rich repeat extends from about amino acid 702 to about aminoacid 727 of SEQ ID NO:8; SEQ ID NO:16. The third Leucine-rich repeatextends from about amino acid 728 to about amino acid 754 of SEQ IDNO:8; SEQ ID NO:17. The fourth Leucine-rich repeat extends from aboutamino acid 755 to about amino acid 782 of SEQ ID NO:8; SEQ ID NO:18. Thefifth Leucine-rich repeat extends from about amino acid 783 to aboutamino acid 810 of SEQ ID NO:8; SEQID NO:19. The sixth Leucine-richrepeat extends from about amino acid 811 to about amino acid 838 of SEQID NO:8: SEQ ID NO:20. The seventh Leucine-rich repeat extends fromabout amino acid 839 to about amino acid 866 of SEQ ID NO:8; SEQ IDNO:21. The eighth Leucine-rich repeat extends from about amino acid 867to about amino acid 894 of SEQ ID NO:8; SEQ ID NO:22. The ninthLeucine-rich repeat extends from about amino acid 895 to about aminoacid 922 of SEQ ID NO:8; SEQ ID NO:23 and the tenth leucine-rich repeatextends from about amino acid 923 to about amino acid 950 of SEQ IDNO:8; SEQ ID NO:24.

The partial cDNA of CARD-4S described below (SEQ ID NO:25) has a 1470nucleotide open reading frame (nucleotides 1-1470 of SEQ ID NO:25; SEQID NO:27) which encodes a 490 amino acid protein (SEQ ID NO:26). CARD-4Sprotein possesses a CARD domain (amino acids 1-74 of SEQ ID NO:26; SEQID NO:28). CARD-4S is predicted to have a P-Loop which extends fromabout amino acid 163 to about amino acid 170 of SEQ ID NO:26; SEQ IDNO:29, and a Walker Box “B” which extends form about amino acid 241 toabout amino acid 245 of SEQ ID NO:26; SEQ ID NO:30.

A human CARD-4Y nucleotide cDNA sequence is presented (SEQ ID NO:38) asis the amino acid sequence of the predicted CARD-4Y product (SEQ IDNO:39). A human CARD-4Z nucleotide cDNA sequence is presented (SEQ IDNO:40) as is the amino acid sequence of the predicted CARD-4Z product(SEQ ID NO:41). A comparison of the CARD-4Y, CARD-4Z, and human CARD-4Lpredicted amino acid sequences is also shown in FIG. 14.

The 761 nucleotide murine CARD-5 cDNA described below (SEQ ID NO:60) hasa 579 nucleotide open reading frame (nucleotides 89 to 668 of SEQ IDNO:60; SEQ ID NO:62) which encodes a 193 amino acid protein (SEQ IDNO:61). Murine CARD-5 contains a CARD domain which extends from aminoacid 110 to amino acid 179 of SEQ ID NO:61 (SEQ ID NO:66).

The 740 nucleotide human CARD-5 cDNA described below (SEQ ID NO:48) hasa 585 nucleotide open reading frame (nucleotides 54 to 639 of SEQ IDNO:48; SEQ ID NO:50) which encodes a 195 amino acid protein (SEQ IDNO:49). Human CARD-5 contains a CARD domain which extends from aminoacid 111 to amino acid 181 of SEQ ID NO:49 (SEQ ID NO:58).

The 5252 nucleotide rat CARD-6 cDNA described below (SEQ ID NO:51) has a2715 nucleotide open reading frame (nucleotides 169 to 2883 of SEQ IDNO:51; SEQ ID NO:53) which encodes a 905 amino acid protein (SEQ IDNO:52). Rat CARD-6 contains a CARD domain which extends from amino acid1 to amino acid 108 of SEQ ID NO:52 (SEQ ID NO:59). Rat CARD-6 also hasa proline-rich C-terminus which extends from amino acid 698 to aminoacid 905 of SEQ ID NO:52 (SEQ ID NO:65). This proline-rich domainincludes five putative SH3 binding sites. These binding sites have thesequence PXXP and are located at amino acids 710 to 713 (PAHP), 806 to809 (PLRP), 819 to 822 (PIPP), 857 to 860 (PPHP), and 881 to 884 (PSQP)of SEQ ID NO:52.

The 4244 nucleotide human CARD-6 cDNA described below (SEQ ID NO:54) hasa 3111 nucleotide open reading frame (nucleotides 200 to 3310 of SEQ IDNO:54; SEQ ID NO:56) which encodes a 1037 amino acid protein (SEQ IDNO:55). Human CARD-6 includes a CARD domain which extends from aminoacid 5 to amino acid 92 of SEQ ID NO:55 (SEQ ID NO:64).

Like other proteins containing a CARD domain, CARD-3, CARD-4, CARD-5,and CARD-6 to participate in the network of interactions that lead tocaspase activity. Human CARD-4L and CARD-5 likely play functional rolesin caspase activation similar to that of Apaf-1 (Zou et al., (1997) Cell90:405-413). For example, upon activation, CARD-4L and CARD-5 binds anucleotide, thus allowing CARD-4L or CARD-5 to bind and activate aCARD-containing caspase via a CARD-CARD interaction, leading toapoptotic death of the cell and/or cytokine processing that leads toinflammation. CARD-3, CARD-4, CARD-5, and CARD-6 molecules are useful asmodulating agents in regulating a variety of cellular processesincluding cell growth and cell death. In one aspect, this inventionprovides isolated nucleic acid molecules encoding CARD-3, CARD-4,CARD-5, or CARD-6 proteins or biologically active portions thereof, aswell as nucleic acid fragments suitable as primers or hybridizationprobes for the detection of CARD-3, CARD-4, CARD-5, or CARD-6 encodingnucleic acids.

The invention encompasses methods of diagnosing and treating patientswho are suffering from a disorder associated with an abnormal level orrate (undesirably high or undesirably low) of apoptotic cell death,abnormal activity of the Fas/APO-1 receptor complex, abnormal activityof the TNF receptor complex, abnormal inflammatory response, or abnormalactivity of a caspase by administering a compound that modulates theexpression of CARD-3, CARD-4, CARD-5, or CARD-6 (at the DNA, mRNA orprotein level, e.g., by altering mRNA splicing) or by altering theactivity of CARD-3, CARD-4, CARD-5, or CARD-6. Examples of suchcompounds include small molecules, antisense nucleic acid molecules,ribozymes, and polypeptides.

Certain disorders are associated with an increased number of survivingcells, which are produced and continue to survive or proliferate whenapoptosis is inhibited or occurs at an undesirably low rate. Compoundsthat modulate the expression or activity of CARD-3, CARD-4, CARD-5, orCARD-6 can be used to treat or diagnose such disorders. These disordersinclude cancer (particularly follicular lymphomas, chronic myelogenousleukemia, melanoma, colon cancer, lung carcinoma, carcinomas associatedwith mutations in p53, and hormone-dependent tumors such as breastcancer, prostate cancer, and ovarian cancer). Such compounds can also beused to treat viral infections (such as those caused by herpesviruses,poxviruses, and adenoviruses). Failure to remove autoimmune cells thatarise during development or that develop as a result of somatic mutationduring an immune response can result in autoimmune disease. Thus,autoimmune disorders can be caused by an undesirably low levels ofapoptosis. Accordingly, modulators of CARD-3, CARD-4, CARD-5, or CARD-6activity or expression can be used to treat autoimmune disorders (e.g.,systemic lupus erythematosis, immune-mediated glomerulonephritis, andarthritis).

Many diseases are associated with an undesirably high rate of apoptosis.Modulators of CARD-3, CARD04, CARD-5, or CARD-6 expression or activitycan be used to treat or diagnose such disorders. For example,populations of cells are often depleted in the event of viral infection,with perhaps the most dramatic example being the cell depletion causedby the human immunodeficiency virus (HIV). Surprisingly, most T cellsthat die during HIV infections do not appear to be infected with HIV.Although a number of explanations have been proposed, recent evidencesuggests that stimulation of the CD4 receptor results in the enhancedsusceptibility of uninfected T cells to undergo apoptosis. A widevariety of neurological diseases are characterized by the gradual lossof specific sets of neurons. Such disorders include Alzheimer's disease,Parkinson's disease. amyotrophic lateral sclerosis (ALS) retinitispigmentosa, spinal muscular atrophy, and various forms of cerebellardegeneration. The cell loss in these diseases does not induce aninflammatory response, and apoptosis appears to be the mechanism of celldeath. In addition, a number of hematologic diseases are associated witha decreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis.

Proteins containing a CARD domain are thought to be involved in variousinflammatory disorders. For example, the CARD domain-containing proteincaspase-1 promotes inflammation by converting certain proinflammatorycytokines from an inactive to an active form. Accordingly, CARD-3,CARD-4, CARD-5, and CARD-6 polypeptides, nucleic acids and modulators ofCARD-3, CARD-4, CARD-5, or CARD-6 expression or activity can be used totreat immune disorders. Such immune disorders include, but are notlimited to, chronic inflammatory diseases and disorders, such as Crohn'sdisease, reactive arthritis, including Lyme disease, insulin-dependentdiabetes, organ-specific autoimmunity, including multiple sclerosis,Hashimoto's thyroiditis and Grave's disease, contact dermatitis,psoriasis, graft rejection, graft versus host disease, sarcoidosis,atopic conditions, such as asthma and allergy, including allergicrhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis), certain viralinfections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

In addition to the aforementioned disorders, CARD-3, CARD-4, CARD-5, andCARD-6 polypeptides, nucleic acids, and modulators of CARD-3, CARD-4,CARD-5 or CARD-6 expression or activity can be used to treat disordersof cell signaling and disorders of tissues in which CARD-3, CARD-4,CARD-5 or CARD-6 is expressed.

The invention features a nucleic acid molecule which is at least 45% (or55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:25,SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:60, SEQ ID NO:62, the nucleotide sequence of the cDNA insertof the plasmid deposited with the ATCC as Accession Number 203037 (the“cDNA of ATCC 203037”), the nucleotide sequence of the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number 203035 (the“cDNA of ATCC 203035”), the nucleotide sequence of the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number 203036 (the“cDNA of ATCC 203036”), the nucleotide sequence of the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number PTA-211 (the“cDNA of ATCC PTA-211”), the nucleotide sequence of the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number PTA-212 (“thecDNA of ATCC PTA-212”), the nucleotide sequence of the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number PTA-213 (the“cDNA of ATCC PTA-213”), or a complement thereof.

The invention features a nucleic acid molecule which includes a fragmentof at least 150 (300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650,700, 800, 900, 1000, 1300, 1600 or 1931) nucleotides of the nucleotidesequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotidesequence of the cDNA ATCC 203037, or a complement thereof.

The invention also features a nucleic acid molecule which includes afragment of at least 150 (350, 400, 450, 500, 550, 600, 650, 700, 800,900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, 3000, or 3382)nucleotides of the nucleotide sequence shown in SEQ ID NO:7, SEQ IDNO:9, or the nucleotide sequence of the cDNA ATCC 203035, or acomplement thereof.

Also within the invention is a nucleic acid molecule which includes afragment of at least 150 (350, 400, 450, 500, 550, 600, 650, 700, 800,900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, and 3080) nucleotides ofthe nucleotide sequence shown in SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:38, SEQ ID NO:40, or the nucleotide sequence of the cDNA of ATCC203036, or a complement thereof.

The invention also features a nucleic acid molecule which includes afragment of at least 150 (350, 400, 450, 500, 550, 600, 650, 700, and761) nucleotides of the nucleotide sequence shown in SEQ ID NO:60, SEQID NO:62, or the nucleotide sequence of the cDNA of ATCC PTA-212, or acomplement thereof.

The invention also features a nucleic acid molecule which includes afragment of at least 150 (350, 400, 450, 500, 550, 600, 650, 700, and740) nucleotides of the nucleotide sequence shown in SEQ ID NO:48, SEQID NO:50, the cDNA of ATCC PTA-213, or a complement thereof.

The invention also features a nucleic acid molecule which includes afragment of at least 150 (350, 400, 450, 500, 600, 700, 800, 900, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, and 5252) nucleotides ofthe nucleotide sequence shown in SEQ ID NO:51, SEQ ID NO:53, or acomplement thereof.

The invention also features a nucleic acid molecule which includes afragment of at least 150 (200, 300, 400, 500, 600, 700, 800, 900, 1000,1400, 1800, 2200, 2600, or 3000) nucleotides of the nucleotide sequenceshown in SEQ ID NO:54, SEQ ID NO:56, the cDNA of ATCC PTA-213, or acomplement thereof.

The invention features a nucleic acid molecule which includes anucleotide sequence encoding a protein having an amino acid sequencethat is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical tothe amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ IDNO:55, SEQ ID NO:61, or the amino acid sequence encoded by the cDNA ofATCC 203037, the amino acid sequence encoded by the cDNA of ATCC 203035,the amino acid sequence encoded by the cDNA of ATCC 203036, the aminoacid sequence encoded by the cDNA of ATCC PTA-211, the amino acidsequence encoded by the cDNA of ATCC PTA-212, or the amino acid sequenceencoded by the cDNA of ATCC PTA-213.

In an embodiment, a CARD-3 nucleic acid molecule has the nucleotidesequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotidesequence of the cDNA of ATCC 203037. In another embodiment, a CARD-4Lnucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:7,or SEQ ID NO:9, or the nucleotide sequence of the cDNA of ATCC 203035.

In yet another embodiment, a CARD-4S nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:25, or SEQ ID NO:27, or thenucleotide sequence of the cDNA of ATCC 203036. In another embodiment, amurine CARD-4L nucleic acid molecule has the nucleotide sequence shownin SEQ ID NO:42.

In another embodiment, a CARD-4Y nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:38.

In another embodiment, a CARD-4Z nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:40.

In another embodiment, a human CARD-5 nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:50 or thenucleotide sequence of the cDNA of ATCC PTA-213. In another embodiment,a murine CARD-5 nucleic acid molecule has the nucleotide sequence shownin SEQ ID NO:60 or SEQ ID NO:62.

In yet another embodiment, a rat CARD-6 nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:51, SEQ ID NO:53, or thenucleotide sequence of the cDNA of ATCC PTA-211.

In still another embodiment, a human CARD-6 nucleic acid molecule hasthe nucleotide sequence shown in SEQ ID NO:54, SEQ ID NO:56, or thenucleotide sequence of the cDNA of ATCC PTA-213.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:2,SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:61, the fragmentincluding at least 15 (25, 30, 50, 100, 150, 300, 400 or 540, 600, 700,800, 900) contiguous amino acids of SEQ ID NO:2, SEQ ID NO:8, SEQ IDNO:26, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ IDNO:52, or SEQ ID NO:55, SEQ ID NO:61, the polypeptide encoded by thecDNA of ATCC Accession Number 203037, the polypeptide encoded by thecDNA of ATCC Accession Number 203035, the polypeptide encoded by thecDNA of ATCC Accession Number 203036, the polypeptide encoded by thecDNA of ATCC Accession Number PTA-211, the polypeptide encoded by thecDNA of ATCC Accession Number PTA-212, or the polypeptide encoded by thecDNA of ATCC Accession Number PTA-213.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ IDNO:61, or an amino acid sequence encoded by the cDNA of ATCC AccessionNumber 203037, 203035, 203036, PTA-211, PTA-212, or PTA-213, wherein thenucleic acid molecule hybridizes to a nucleic acid molecule consistingof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:25, SEQ IDNO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:60, SEQ ID NO:62, the cDNA of ATCC 203037, the cDNA of ATCC 203035,the cDNA of ATCC 203036, the cDNA of ATCC PTA-211, the cDNA of ATCCPTA-212, or the cDNA of PTA-213 under stringent conditions.

In general, an allelic variant of a gene will be readily identifiable asmapping to the same chromosomal location as said gene. For example, inExample 6, the clromosomal location of the human CARD-4 gene isdiscovered to be chromosome 7 close to the SHGC-31928 genetic marker.Allelic variants of human CARD-4 will be readily identifiable as mappingto the human CARD-4 locus on chromosome 7 near genetic markerSHGC-31928.

Also within the invention are: an isolated CARD-3 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO:2; anisolated CARD-3 protein having an amino acid sequence that is at leastabout 85%, 95%, or 98% identical to the kinase domain of SEQ ID NO:2(e.g., about amino acid residues 1 to 300 of SEQ ID NO:2; SEQ ID NO:4);and an isolated CARD-3 protein having an amino acid sequence that is atleast about 85%, 95%, or 98% identical to the linker domain of SEQ IDNO:2 (e.g., about amino acid residues 301 to 431 of SEQ ID NO:2; SEQ IDNO:5); an isolated CARD-3 protein having an amino acid sequence that isat least about 85%, 95%, or 98% identical to the CARD domain of SEQ IDNO:2 (e.g., about amino acid residues 432 to 540 of SEQ ID NO:2; SEQ IDNO:6); an isolated CARD-4L protein having an amino acid sequence that isat least about 65%, preferably 75%, 85%, 95%, or 98% identical to theamino acid sequence of SEQ ID NO:8; an isolated CARD-4L protein havingan amino acid sequence that is at least about 85%, 95%, or 98% identicalto the CARD domain of SEQ ID NO:8 (e.g., about amino acid residues 15 to114 of SEQ ID NO:8; SEQ ID NO:10); an isolated CARD-4L protein having anamino acid sequence that is at least about 85%, 95%, or 98% identical tothe nucleotide binding domain of SEQ ID NO:8 (e.g., about amino acidresidues 198 to 397 of SEQ ID NO:8; SEQ ID NO:11; an isolated CARD-4Lprotein having an amino acid sequence that is at least about 85%, 95%,or 98% identical to the kinase 1a (P-loop) subdomain SEQ ID NO:8 (e.g.,about amino acid 127 to about amino acid 212 of SEQ ID NO:8; SEQ IDNO:46); an isolated CARD-4L protein having an amino acid sequence thatis at least about 85%, 95%, or 98% identical to the kinase 2 subdomainof SEQ ID NO:8 (e.g., about amino acid 273 to about amino acid 288 ofSEQ ID NO:8: SEQ ID NO:47); an isolated CARD-4L protein having an aminoacid sequence that is at least about 85%, 95%, or 98% identical to akinase 3a subdomain of SEQ ID NO:8 (e.g., about amino acid residues 327to 338 of SEQ ID NO:8; SEQ ID NO:14); an isolated CARD-4L protein havingan amino acid sequence that is at least about 85%, 95%, or 98% identicalto the Leucine-rich repeats of SEQ ID NO:8 (e.g., about amino acidresidues 674 to 701 of SEQ ID NO:8; SEQ ID NO:15; from amino acid 702 toamino acid 727 of SEQ ID NO:8; SEQ ID NO:16; which extends from aminoacid 728 to amino acid 754 SEQ ID NO:8; SEQ ID NO:17; from amino acid755 to amino acid 782 of SEQ ID NO:8; SEQ ID NO:18; from amino acid 783to amino acid 810 of SEQ ID NO:8; SEQ ID NO:19; from amino acid 811 toamino acid 838 of SEQ ID NO:8; SEQ ID NO:20 from amino acid 839 to aminoacid 866 of SEQ ID NO:8; SEQ ID NO:21; from amino acid 867 to amino acid894 of SEQ ID NO:8; SEQ ID NO:22; from amino acid 895 to amino acid 922of SEQ ID NO:8; SEQ ID NO:23; and from amino acid 923 to amino acid 950of SEQ ID NO:8; SEQ ID NO:24); an isolated CARD-4S protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO:26; anisolated CARD-4S protein having an amino acid sequence that is at leastabout 85%, 95%, or 98% identical to the CARD domain of SEQ ID NO:26(e.g., about amino acid residues 1 to 74 of SEQ ID NO:26; SEQ ID NO:28).Also within the invention are: an isolated murine CARD-4L protein havingan amino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO:43. Alsowithin the invention arean isolated CARD-4Y protein having an amino acidsequence that is at least about 65%, preferably 75%, 85%, 95%, or 98%identical to the amino acid sequence of SEQ ID NO:39. Also within theinvention are: an isolated CARD-4Z protein having an amino acid sequencethat is at least about 65%, preferably 75%, 85%, 95%, or 98% identicalto the amino acid sequence of SEQ ID NO:41.

Also within the invention are: an isolated CARD-5 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO:49 and anisolated CARD-5 protein comprising an amino acid sequence that is atleast about 90%, 95%, or 98% identical to SEQ ID NO:58 (CARD domain).

Also within the invention are an isolated CARD-5 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the amino acid sequence of SEQ ID NO:60 and an isolatedCARD-5 protein comprising an amino acid sequence that is at least about90%, 95%, or 98% identical to SEQ ID NO:57 (CARD domain).

The invention also includes: an isolated CARD-6 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the amino acid sequence of SEQ ID NO:52 and an isolatedCARD-6 protein having an amino acid sequence that is at least about 90%,95%, or 98% identical to SEQ ID NO:59 (CARD domain).

The invention also includes: an isolated CARD-6 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the amino acid sequence of SEQ ID NO:55 and an isolatedCARD-6 protein having an amino acid sequence that is at least about 90%,95%, or 98% identical to SEQ ID NO:64 (CARD domain).

Also within the invention are: an isolated CARD-3 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:3or the cDNA of ATCC 203037; an isolated CARD-3 protein which is encodedby a nucleic acid molecule having a nucleotide sequence at least about65% preferably 75%, 85%, or 95% identical to the kinase domain encodingportion of SEQ ID NO:1 (e.g., about nucleotides 213 to 1113 of SEQ IDNO:1); an isolated CARD-3 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical the linker domain encoding portion of SEQ ID NO:1(e.g., about nucleotides 1114 to 1506 of SEQ ID NO:1); and an isolatedCARD-3 protein which is encoded by a nucleic acid molecule having anucleotide sequence at least about 65% preferably 75%, 85%, or 95%identical the CARD domain encoding portion of SEQ ID NO:1 (e.g., aboutnucleotides 1507 to 1833 of SEQ ID NO:1); and an isolated CARD-3 proteinwhich is encoded by a nucleic acid molecule having a nucleotide sequencewhich hybridizes under stringent hybridization conditions to a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:3 or thenon-coding strand of the cDNA of ATCC 203037. Also within the inventionare: an isolated CARD-4Y protein which is encoded by a nucleic acidmolecule having a nucleotide sequence that is at least about 65%,preferably 75%, 85%, or 95% identical to SEQ ID NO:38. Also within theinvention are nucleic acid molecules which include about nucleotides2759 to 2842 of SEQ ID NO:7; about nucleotides 2843 to 2926 of SEQ IDNO:7; about nucleotides 2927 to 3010 of SEQ ID NO:7; about nucleotides3011 to 3094 of SEQ ID NO:7; and an isolated CARD-4L protein which isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:9, or thenon-coding strand of the cDNA of ATCC 203035.

Also within the invention are an isolated CARD-4S protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:27; an isolated CARD-3 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical the CARD domain encoding portion of SEQ ID NO:25(e.g., about nucleotides 1 to 222 of SEQ ID NO:25); an isolated CARD-3protein which is encoded by a nucleic acid molecule having a nucleotidesequence at least about 65% preferably 75%, 85%, or 95% identical theP-Loop encoding portion of SEQ ID NO:25 (e.g., about nucleotides 485 to510 of SEQ ID NO:25).

Also within the invention are an isolated CARD-5 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:48 or the cDNA of ATCC PTA-213; an isolated CARD-5 protein which isencoded by a nucleic acid molecule having a nucleotide sequence at leastabout 90% preferably 95%, or 98% identical to the CARD encoding portionof SEQ ID NO:48 (e.g., about nucleotides 383 to 596 of SEQ ID NO:48);and an isolated CARD-5 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:48 or the non-coding strand of the cDNAof ATCC PTA-213.

Also within the invention are an isolated CARD-5 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:60; an isolated CARD-5 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 90% preferably 95%,or 98% identical to the CARD encoding portion of SEQ ID NO:60 (e.g.,about nucleotides 416 to 625 of SEQ ID NO:60); and an isolated CARD-5protein which is encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule having the nucleotide sequence of SEQ ID NO:60.

Also within the invention are an isolated CARD-6 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:51; an isolated CARD-6 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 90% preferably 95%,or 98% identical to the CARD encoding portion of SEQ ID NO:51 (e.g.,about nucleotides 169 to 456 of SEQ ID NO:51); and an isolated CARD-6protein which is encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule having the nucleotide sequence of SEQ ID NO:51.

Also within the invention are an isolated CARD-6 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:54; an isolated CARD-6 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 90% preferably 95%,or 98% identical to the CARD encoding portion of SEQ ID NO:54; and anisolated CARD-6 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:54.

Another embodiment of the invention features CARD-3, CARD-4, CARD-5, orCARD-6 nucleic acid molecules which specifically detect CARD-3, CARD-4,CARD-5, or CARD-6 nucleic acid molecules, relative to nucleic acidmolecules encoding other members of the CARD superfamily. For example,in one embodiment, a CARD-4L nucleic acid molecule hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, or the cDNA of ATCC203035, or a complement thereof. In another embodiment, the CARD-4Lnucleic acid molecule is at least 300 (350, 400, 450, 500, 550, 600,650, 700, 800, 900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, 3000, or3382) nucleotides in length and hybridizes under stringent conditions toa nucleic acid molecule comprising the nucleotide sequence shown in SEQID NO:7, SEQ ID NO:9, the cDNA of ATCC 203035, or a complement thereof.In another embodiment, an isolated CARD-4L nucleic acid moleculecomprises nucleotides 287 to 586 of SEQ ID NO:7, encoding the CARDdomain of CARD-4L, or a complement thereof. In yet another embodiment,the invention provides an isolated nucleic acid molecule which isantisense to the coding strand of a CARD-4L nucleic acid.

In another embodiment, a CARD-5 nucleic acid molecule hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:48, SEQ ID NO:50, or the cDNA of ATCCPTA-213, or a complement thereof. In another embodiment, the CARD-5nucleic acid molecule is at least 300 (350, 400, 450, 500, 550, 585,600, 650, 700, or 740) nucleotides in length and hybridizes Linderstringent conditions to a nucleic acid molecule comprising thenucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:50, the cDNA ofATCC PTA-213, or a complement thereof. In another embodiment, anisolated CARD-5 nucleic acid molecule comprises nucleotides 383 to 596of SEQ ID NO:48, encoding the CARD of CARD-5. In yet another embodiment,the invention provides an isolated nucleic acid molecule which isantisense to the coding strand of a CARD-5 nucleic acid.

Another aspect of the invention provides a vector, e.g., a recombinantexpression vector, comprising a CARD-3, CARD-4, CARD-5, or CARD-6nucleic acid molecule of the invention. In another embodiment theinvention provides a host cell containing such a vector. The inventionalso provides a method for producing CARD-3, CARD-4, CARD-5, or CARD-6protein by culturing, in a suitable medium a host cell of the inventioncontaining a recombinant expression vector such that a CARD-3, CARD-4,CARD-5, or CARD-6 protein is produced.

Another aspect of this invention features isolated or recombinantCARD-3, CARD-4, CARD-5, or CARD-6 proteins and polypeptides. PreferredCARD-3, CARD-4, CARD-5, or CARD-6 proteins and polypeptides possess atleast one biological activity possessed by naturally occurring humanCARD-3, CARD-4, CARD-5, or CARD-6, e.g., (1) the ability to formprotein:protein interactions with proteins in the apoptotic signalingpathway: (2) the ability to form CARD-CARD interactions with proteins inthe apoptotic signaling pathway, e.g., caspase-1; (3) the ability tobind a CARD-3, CARD-4, CARD-5, or CARD-6 ligand; and (4) the ability tobind to an intracellular target. Other activities include: (1)modulation of cellular proliferation; (2) modulation of cellulardifferentiation; (3) modulation of cellular death; (4) modulation of theNF-κB pathway; (5) modulation of proinflammatory cytokine activation;and (6) modulation of inflammation.

The CARD-3, CARD-4, CARD-5, or CARD-6 proteins of the present inventionor biologically active portions thereof can be operatively linked to anon-CARD-3, non-CARD-4, non-CARD-5, or non-CARD-6 polypeptide (e.g.,heterologous amino acid sequences) to form CARD-3, CARD-4, CARD-5, orCARD-6 fusion proteins respectively. The invention further featuresantibodies that specifically bind CARD-3, CARD-4, CARD-5, or CARD-6proteins, such as monoclonal or polyclonal antibodies. In addition, theCARD-3, CARD-4, CARD-5, or CARD-6 proteins or biologically activeportions thereof can be incorporated into pharmaceutical compositions,which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of CARD-3, CARD-4, CARD-5, or CARD-6 activity or expressionin a biological sample by contacting the biological sample with an agentcapable of detecting an indicator of CARD-3, CARD-4, CARD-5, or CARD-6activity such that the presence of CARD-3, CARD-4, CARD-5, or CARD-6activity is detected in the biological sample.

In another aspect, the invention provides a method for modulatingCARD-3, CARD-4, CARD-5, or CARD-6 activity comprising contacting a cellwith an agent that modulates (inhibits or stimulates) CARD-3, CARD-4,CARD-5, or CARD-6 activity or expression such that CARD-3, CARD-4,CARD-5, or CARD-6 activity or expression in the cell is modulated. Inone embodiment, the agent is an antibody that specifically binds toCARD-3, CARD-4, CARD-5, or CARD-6 protein. In another embodiment, theagent modulates expression of CARD-3, CARD-4, CARD-5, or CARD-6 bymodulating transcription of a CARD-3, CARD-4, CARD-5, or CARD-6 gene,splicing of a CARD-3, CARD-4, CARD-5, or CARD-6 mRNA, or translation ofa CARD-3, CARD-4, CARD-5, or CARD-6 mRNA. In yet another embodiment, theagent is a nucleic acid molecule having a nucleotide sequence that isantisense to the coding strand of the CARD-3, CARD-4, CARD-5, or CARD-6mRNA or the CARD-3, CARD-4, CARD-5, or CARD-6 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant CARD-3,CARD-4, CARD-5, or CARD-6 protein or nucleic acid expression or activityor related to CARD-3, CARD-4, CARD-5, or CARD-6 expression or activityby administering an agent which is a CARD-3, CARD-4, CARD-5, or CARD-6modulator to the subject. In one embodiment, the CARD-3, CARD-4, CARD-5,or CARD-6 modulator is a CARD-3, CARD-4, CARD-5, or CARD-6 protein. Inanother embodiment the CARD-3, CARD-4, CARD-5, or CARD-6 modulator is aCARD-3, CARD-4, CARD-5, or CARD-6 nucleic acid molecule. In otherembodiments, the CARD-3, CARD-4, CARD-5, or CARD-6 modulator is apeptide, peptidomimetic, or other small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a CARD-3, CARD-4, CARD-5, or CARD-6 protein; (ii)mis-regulation of a gene encoding a CARD-3, CARD-4, CARD-5, or CARD-6protein; (iii) aberrant RNA splicing; and (iv) aberrantpost-translational modification of a CARD-3, CARD-4, CARD-5, or CARD-6protein, wherein a wild-type form of the gene encodes a protein with aCARD-3, CARD-4, CARD-5, or CARD-6 activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a CARD-3, CARD-4,CARD-5, or CARD-6 protein. In general, such methods entail measuring abiological activity of a CARD-3, CARD-4, CARD-5, or CARD-6 protein inthe presence and absence of a test compound and identifying thosecompounds which alter the activity of the CARD-3, CARD-4, CARD-5, orCARD-6 protein.

The invention also features assays for identifying compounds that blockthe interaction of CARD-5 with a CARD-5 ligand, e.g., caspase-1, CARD-7,or CARD-5. Isolated CARD domains of CARD-5, caspase-1 and/or CARD-7 canbe used in these assays.

The invention also features methods for identifying a compound whichmodulates the expression of CARD-3, CARD-4, CARD-5, or CARD-6 bymeasuring the expression of CARD-3, CARD-4, CARD-5, or CARD-6 in thepresence and absence of a compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA sequence (SEQ ID NO:1) of human CARD-3. The openreading frame of CARD-3 (SEQ ID NO:1) extends from nucleotide 213 tonucleotide 18833 nucleotide (SEQ ID NO:3).

FIG. 2 depicts the predicted amino acid sequence (SEQ ID NO:2) of humanCARD-3.

FIG. 3 depicts the cDNA sequence (SEQ ID NO:7) of CARD-4L. The openreading frame of SEQ ID NO:7 extends from nucleotide 245 to nucleotide3103 (SEQ ID NO:9).

FIG. 4 depicts the predicted amino acid sequence (SEQ ID NO:8) of humanCARD-4L.

FIG. 5 depicts the partial cDNA sequence (SEQ ID NO:25) of CARD-4S andthe predicted amino acid sequence (SEQ ID NO:25) of human CARD-4S. Theopen reading frame of CARD-4 (SEQ ID NO:25) extends from nucleotide 1 tonucleotide 1470 (SEQ ID NO:27).

FIG. 6 depicts the predicted amino acid sequence (SEQ ID NO:26) of humanCARD-4S.

FIG. 7 depicts an alignment of the CARD domains of CARD-4 (SEQ IDNO:10), CARD-3 (SEQ ID NO:6), ARC-CARD (SEQ ID NO:31), cIAP1-CARD (SEQID NO:32), and cIAP2-CARD (SEQ ID NO:33).

FIG. 8 is a plot showing predicted structural features of human CARD-4L.

FIG. 9 is a plot showing predicted structural features of human CARD-4S.

FIG. 10 depicts the cDNA sequence (SEQ ID NO:38) of the human CARD-4Ysplice variant clone. The predicted open reading frame of the humanCARD-4Y splice variant clone extends from nucleotide 438 to nucleotide1184.

FIG. 11 depicts the amino acid sequence (SEQ ID NO:39) of the proteinpredicted to be encoded by the human CARD-4Y cDNA open reading frame.

FIG. 12 depicts the cDNA sequence (SEQ ID NO:40) of the human CARD-4Zsplice variant clone. The predicted open reading frame of the humanCARD-4Z splice variant clone extends from nucleotide 489 to nucleotide980.

FIG. 13 depicts the amino acid sequence (SEQ ID NO:41) ofthe proteinpredicted to be encoded by the human CARD-4Z cDNA open reading frame.

FIG. 14 depicts an alignment of human CARD-4L (SEQ ID NO:8), thepredicted amino acid sequence of human CARD-4Y (SEQ ID NO:39), and thepredicted amino acid sequence of human CARD-4Z (SEQ ID NO:41).

FIG. 15 depicts the nucleotide sequence of the murine CARD-4L cDNA (SEQID NO:42).

FIG. 16 depicts the predicted amino acid sequence of murine CARD-4L (SEQID NO:43).

FIG. 17 depicts an alignment of human CARD-4L (SEQ ID NO:8) and thepredicted amino acid sequence of murine CARD-4L (SEQ ID NO:43).

FIG. 18 depicts a 32042 nucleotide genomic sequence of CARD-4.

FIG. 19 depicts the nucleotide sequence of a murine CARD-5 cDNA (SEQ IDNO:60). The open reading frame of this cDNA extends from nucleotide 89to nucleotide 667 of SEQ ID NO:60 (SEQ ID NO:62) and encodes a 193 aminoacid protein (SEQ ID NO:61).

FIG. 20 depicts a hydropathy plot of murine CARD-5. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and potential N-glycosylation sites (Ngly)are indicated by short vertical lines just below the hydropathy trace.

FIG. 21 depicts the nucleotide sequence of a human CARD-5 cDNA (SEQ IDNO:48). The open reading frame of this cDNA extends from nucleotide 53to nucleotide 638 of SEQ ID NO:48 (SEQ ID NO:50) and encodes a 195 aminoacid protein SEQ ID NO:49).

FIG. 22 depicts a hydropathy plot of human CARD-5. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and potential N-glycosylation sites (Ngly)are indicated by short vertical lines just below the hydropathy trace.

FIG. 23 depicts an alignment of the cDNA sequences of murine CARD-5 (SEQID NO:60) and human CARD-5 (SEQ ID NO:48). This alignment was createdusing ALIGN (version 2.0, PAM120 scoring matrix; −12/−4 gap penalty). Inthis alignment the sequences are 68.2% identical.

FIG. 24 depicts an alignment of the amino acid sequences of murineCARD-5 (SEQ ID NO:61) and human CARD-5 (SEQ ID NO:49). This alignmentwas created using ALIGN (version 2.0, PAM120 scoring matrix; −12/−4 gappenalty). In this alignment the sequences are 71.8% identical.

FIG. 25 depicts the nucleotide sequence of a rat CARD-6 cDNA (SEQ IDNO:51). The open reading frame of this cDNA extends from nucleotide 169to nucleotide 2883 of SEQ ID NO:51 (SEQ ID NO:53) and encodes a 505amino acid protein (SEQ ID NO:52).

FIG. 26 depicts a hydropathy plot of rat CARD-6. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and potential N-glycosylation sites (Ngly) are indicatedby short vertical lines just below the hydropathy trace.

FIG. 27 depicts an alignment of the CARD domains of murine CARD-5 (SEQID NO:57), human CARD-5 (SEQ ID NO:58), and RAIDD (SEQ ID NO:70).

FIG. 28 depicts the nucleotide sequence of a human CARD-6 cDNA (SEQ IDNO:54). The open reading frame of this cDNA extends from nucleotide 200to 3310 of SEQ ID NO:54 (SEQ ID NO:56) and encodes a 1037 amino acidprotein (SEQ ID NO:55).

FIG. 29 depicts a hydropathy plot of human CARD-6. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.

FIG. 30 depicts an alignment of the CARD domain of human CARD-6 (SEQ IDNO:64) with a consensus CARD domain (SEQ ID NO:67). In this depiction ofthe consensus sequence, more conserved residues are indicated byuppercase letters and less conserved residues are indicated by lowercaseletters.

FIG. 31 depicts an alignment of the CARD domains of human CARD-3, humanCARD-4, human CARD-5, murine CARD-5, human CARD-6, and rat CARD-6. Thisalignment was created using the Clustal method with PAM250 residueweight table. A consensus sequence is also depicted (SEQ ID NO:71).

DETAILED FESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of cDNAmolecules encoding human CARD-3, human CARD-4, partial murine CARD-4L,murine CARD-5, human CARD-5, rat CARD-6, and human CARD-6 proteins.TABLE 1 Summary of CARD-3. CARD-4. CARD-5, and CARD-6 SequenceInformation. Accession Gene cDNA Protein ORF FIGURE Number human SEQ IDSEQ ID SEQ ID FIGS. 203037 CARD-3 NO:1 NO:2 NO:3 1-2 human SEQ ID SEQ IDSEQ ID FIGS. 203035 CARD-4L NO:7 NO:8 NO:9 3-4 human SEQ ID SEQ ID SEQID FIGS. 203036 CARD-4S NO:25 NO:26 NO:27 5-6 human SEQ ID SEQ ID FIGS.CARD-4Y NO:38 NO:39 10-11 human SEQ ID SEQ ID FIGS. CARD-4Z NO:40 NO:4112-13 murine SEQ ID SEQ ID FIGS. CARD-4L NO:42 NO:43 15-16 human SEQ IDSEQ ID SEQ ID PTA-213 CARD-5 NO:48 NO:49 NO:50 murine SEQ ID SEQ ID SEQID PTA-212 CARD-5 NO:60 NO:61 NO:62 human SEQ ID SEQ ID SEQ ID PTA-213CARD-6 NO:54 NO:55 NO:56 rat SEQ ID SEQ ID SEQ ID PTA-211 CARD-6 NO:51NO:52 NO:53

A nucleotide sequence encoding a human CARD-3 protein is shown in FIG. 1(SEQ ID NO:1; SEQ ID NO:3 includes the open reading frame only). Apredicted amino acid sequence of CARD-3 protein is also shown in FIG. 2(SEQ ID NO:2).

CARD-4 has at least two forms, a long form, CARD-4L, and a short form,CARD-4S as well as two or more splice variants. A nucleotide sequenceencoding a human CARD-4L protein is shown in FIG. 3 (SEQ ID NO:7; SEQ IDNO:9 includes the open reading frame only). A predicted amino acidsequence of CARD-4L protein is also shown in FIG. 4 (SEQ ID NO:8). Anucleotide sequence encoding a human CARD-4S protein is shown in FIG. 5(SEQ ID NO:25; SEQ ID NO:27 includes the open reading frame only). Apredicted amino acid sequence of CARD-4S protein is shown in FIG. 6 (SEQID NO:26). Two additional splice variants of human CARD-4 are providedin FIGS. 10 and 11 (human CARD-4Y) and FIGS. 12 and 13 (human CARD-4Z)(predicted amino acid sequences: SEQ ID NO:39 and SEQ ID NO:41 andnucleic acid sequences: SEQ ID NO:38 and SEQ ID NO:40). These two splicevariants are predicted to contain 249 and 164 amino acids, respectively.An alignment of human CARD-4Y, human CARD-4Z and human CARD-4L is shownin FIG. 14.

In addition to the human CARD-4 proteins, a full length nucleotidesequence of the murine ortholog of human CARD-4L is provided in FIG. 15(SEQ ID NO:42). An alignment of murine CARD-4L with human CARD-4L isshown in FIG. 17.

A nucleotide sequence encoding a murine CARD-5 protein is shown in FIG.19 (SEQ ID NO:60; SEQ ID NO:62 includes the open reading frame only). Apredicted amino acid sequence of murine CARD-5 protein is also shown inFIG. 19 (SEQ ID NO:61).

A nucleotide sequence encoding a human CARD-5 protein is shown in FIG.21 (SEQ ID NO:48: SEQ ID NO:50 includes the open reading frame only). Apredicted amino acid sequence of human CARD-5 protein is also shown inFIG. 21 (SEQ ID NO:49).

A nucleotide sequence encoding a rat CARD-6 protein is shown in FIG. 25(SEQ ID NO:51; SEQ ID NO:53 includes the open reading frame only). Apredicted amino acid sequence of rat CARD-6 protein is also shown inFIG. 25 (SEQ ID NO:52).

The human CARD-3 cDNA of FIG. 1 (SEQ ID NO:1), which is approximately1931 nucleotides long including untranslated regions. encodes a proteinhaving a molecular weight of approximately 61 kDa (excludingpost-translational modifications).

A plasmid containing a cDNA encoding human CARD-3 (pXE17A) was depositedwith the American Type Culture Collection (ATCC), Manasass, Va. on May14, 1998, and assigned Accession Number 203037. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

The human CARD-4L cDNA of FIG. 3 (SEQ ID NO:7), which is approximately3382 nucleotides long including untranslated regions encodes a proteinhaving a molecular weight of approximately 108 kDa (excludingpost-translational modifications).

A plasmid containing a cDNA encoding human CARD-4L (pC4LI) was depositedwith the American Type Culture Collection (ATCC), Manasass, Va. on Jul.7, 1998, and assigned Accession Number 203035. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

The human CARD-4S cDNA of FIG. 5 (SEQ ID NO:25), which is 3082nucleotides long including untranslated regions.

A plasmid containing a cDNA encoding human CARD-4S (pDB33E) wasdeposited with the American Type Culture Collection (ATCC), Manasass,Va. on May 14, 1998, and assigned Accession Number 203036. This depositwill be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The human CARD-5 cDNA of FIG. 21 (SEQ ID NO:48), which is approximately740 nucleotides long including untranslated regions, encodes a proteinhaving a molecular weight of approximately 21.6 kD.

A plasmid containing a cDNA encoding human CARD-5 (EpHC5) was depositedwith the American Type Culture Collection (ATCC), Manasass, Va. on Jun.11, 1999, and assigned Accession Number PTA-213. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

The murine CARD-5 cDNA of FIG. 19 (SEQ ID NO:60), which is approximately778 nucleotides long, including untranslated regions, encodes a proteinhaving a molecular weight of approximately 21.5 kD.

A plasmid containing a cDNA encoding murine CARD-5 (EpMC5) was depositedwith the American Type Culture Collection (ATCC), Manassas, Va. on Jun.11, 1999, and assigned Accession Number PTA-212. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

The human CARD-6 cDNA of FIG. 28 (SEQ ID NO:54), which is approximately4244 nucleotides long encodes a protein having a molecular weight ofapproximately 116.5 kD (excluding post-translational modifications).

A plasmid containing a cDNA encoding an amino terminal portion of humanCARD-6 (EpHC6e), a plasmid encoding a carboxy terminal portion of humanCARD-6 (EpHC6c), and a plasmid containing cDNA encoding human CARD-6(EpHC6) were deposited with the American Type Culture Collection (ATCC),Manasass, Va. on Jun. 11, 1999, and assigned Accession Number PTA-213.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The rat CARD-6 cDNA of FIG. 25 (SEQ ID NO:51), which is approximately5252 nucleotides long including untranslated regions, encodes a proteinhaving a molecular weight of approximately 100.7 kD.

A plasmid containing a cDNA encoding rat CARD-6 (EpMR5) was depositedvith the American Type Culture Collection (ATCC), Manassas, Va. on Jun.10, 1999, and assigned Accession Number PTA-211. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

A region of human CARD-4L protein (SEQ ID NO:8), the CARD domain (SEQ IDNO:10), bears some similarity to a CARD domain of CARD-3 (SEQ ID NO:6),ARC-CARD (SEQ ID NO:31), cIAP1-CARD (SEQ ID NO:32), and cIAP2-CARD (SEQID NO:33). This comparison is depicted in FIG. 7.

A region, the CARD domain (SEQ ID NO:58), of human CARD-5 protein (SEQID NO:48) and a region, the CARD domain (SEQ ID NO:57), of murine CARD-5protein (SEQ ID NO:61) bear some similarity to the CARD of RAIDD (SEQ IDNO:70). This comparison is depicted in FIG. 27.

Each of CARD-3, CARD-4, CARD-5, and CARD-6 are members of a family ofmolecules (the “CARD-3 family”, the “CARD-4 family”, the “CARD-5family”, and the “CARD-6 family” respectively) having certain conservedstructural and functional features. The term “family” when referring tothe protein and nucleic acid molecules of the invention is intended tomean two or more proteins or nucleic acid molecules having a commonstructural domain and having sufficient amino acid or nucleotidesequence identity as defined herein. Such family members can benaturally occurring and can be from either the same or differentspecies. For example, a family can contain a first protein of humanorigin and a homologue of that protein of murine origin, as well as asecond, distinct protein of human origin and a murine homologue of thatprotein. Members of a family may also have common functionalcharacteristics.

In one embodiment, a CARD-3, CARD-4, CARD-5, or CARD-6 protein includesa CARD domain having at least about 65%, preferably at least about 75%,and more preferably about 85%, 95%, or 98% amino acid sequence identityto the CARD domain of SEQ ID NO:6 or the CARD domain of SEQ ID NO:10 orthe CARD domain of SEQ ID NO:28, the CARD domain of SEQ ID NO:57, theCARD domain of SEQ ID NO:58, the CARD domain of SEQ ID NO:59, or theCARD domain of SEQ ID NO:64.

Preferred CARD-3, CARD-4, CARD-5, or CARD-6 polypeptides of the presentinvention have an amino acid sequence sufficiently identical to the CARDdomain amino acid sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:57,SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:64, respectively.

The CARD-3 polypeptide also has an amino acid sequence sufficientlyidentical to the kinase domain sequence of SEQ ID NO:4, and an aminoacid sequence that is sufficiently identical to the linker domain of SEQID NO:5. The CARD-4L polypeptide has an amino acid sequence sufficientlyidentical to the nucleotide binding domain of SEQ ID NO:11 an amino acidsequence sufficiently identical to the Walker Box “A” of SEQ ID NO:12 orWalker Box “B” of SEQ ID NO:13, an amino acid sequence sufficientlyidentical to the kinase 1a subdomain of SEQ ID NO:46 an amino acidsequence sufficiently identical to the kinase 2 subdomain of SEQ IDNO:47, or an amino acid sequence sufficiently identical to the kinase 3asubdomain of SEQ ID NO:14, or an amino acid sequence sufficientlyidentical to the Leucine-rich repeats of SEQ ID NO:15, SEQ ID NO:16, SEQID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, and SEQ ID NO:24.

As used herein, the term “sufficiently identical” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain) amino acid residues or nucleotides to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences have a common structural domain and/orcommon functional activity. For example, amino acid or nucleotidesequences which contain a common structural domain having about 65%identity, preferably 75% identity, more preferably 85%, 95%, or 98%identity are defined herein as sufficiently identical.

As used interchangeably herein a “CARD-3, CARD-4, CARD-5, or CARD-6activity”, “biological activity of CARD-3, CARD-4, CARD-5, or CARD-6” or“functional activity of CARD-3, CARD-4, CARD-5, or CARD-6”, refers to anactivity exerted by a CARD-3, CARD-4, CARD-5, or CARD-6 protein,polypeptide or nucleic acid molecule on a CARD-3, CARD-4, CARD-5, orCARD-6 responsive cell as determined in vivo, or in vitro, according tostandard techniques. A CARD-3, CARD-4, CARD-5, or CARD-6 activity can bea direct activity, such as an association with or an enzymatic activityon a second protein or an indirect activity, such as a cellularsignaling activity mediated by interaction of the CARD-3, CARD-4,CARD-5, or CARD-6 protein with a second protein. In one embodiment, aCARD-3, CARD-4, CARD-5, or CARD-6 activity includes at least one or moreof the following activities: (i) the ability to interact with proteinsin an apoptotic signaling pathway (ii) the ability to interact with aCARD-3, CARD-4, CARD-5, or CARD-6 ligand; (iii) the ability to interactwith an intracellular target protein; (iv) the ability to interact,directly or indirectly with one or more with caspases, e.g., caspase-1;(v) the ability to modulate the activity of a caspase, e.g., caspase-9;(vi) the ability to modulate the activity of NF-κB; (vii) the ability tomodulate Apaf-1; (viii) the ability to be modulated by a Bcl-2 familymember; (ix) the ability to be modulated by the p75 neurotrophinreceptor; and (x) the ability to modulate the activity of a stressactivated kinase (e.g., JNK/p38). For example, in Example 4,CARD-3-containing proteins were shown to associate withCARD-4-containing proteins. In example 9, CARD-4 proteins were shown toinduce NF-κB-mediated transcription. In example 10, CARD-3 and CARD-4were shown to enhance caspase-9 activity. In example 17, CARD-5 wasshown to bind the CARD domains of caspase-1, CARD-7, and CARD-5. Inexample 18, CARD-5 was shown to induce NF-κB-mediated transcription.

CARD-5 may modulate caspase-1 activation by binding to caspase-1.CARD-5, binding to caspase-1 may effect an oligomerizaton-basedactivation of the molecule. This caspase-1 activation can lead to theactivation of inflammatory and/or apoptotic pathways. Alternatively,CARD-5 binding of caspase-1 may attenuate caspase-1 activation mediatedby other activator adaptors such as RIP2.

CARD-3 may bind to the p75 neurotrophin receptor. CARD-3 may transducethe activity of the p75 neurotrophin receptor and thus modulateapoptosis of neuronal cells. Accordingly, CARD-3 nucleic acids andpolypeptides as well as modulators of CARD-3 activity or expression canbe used to modulate apoptosis of neurons (e.g., for treatment ofneurological disorders, particularly neurodegenerative disorders).

Accordingly, another embodiment of the invention features isolatedCARD-3, CARD-4, CARD-5, or CARD-6 proteins and polypeptides having aCARD-3, CARD-4, CARD-5, or CARD-6 activity.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode CARD-3, CARD-4, CARD-5, or CARD-6 proteins or biologicallyactive portions thereof, as well as nucleic acid molecules sufficientfor use as hybridization probes to identify CARD-3, CARD-4, CARD-5, orCARD-6-encoding nucleic acids (e.g., CARD-3, CARD-4, CARD-5, or CARD-6mRNA) and fragments for use as PCR primers for the amplification ormutation of CARD-3, CARD-4, CARD-5, or CARD-6 nucleic acid molecules. Asused herein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences (preferably protein encoding sequences) which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatedCARD-3, CARD-4, CARD-5, or CARD-6 nucleic acid molecule can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material or culture medium whenproduced by recombinant techniques or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:7, SEQ ID NO:9, SEQ ID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, the cDNAof ATCC 203037, the cDNA of ATCC 203035, the cDNA of ATCC 203036, thecDNA of ATCC PTA-211, the cDNA of ATCC PTA-212, or the cDNA of ATCCPTA-213, or a complement of any of these nucleotide sequences, can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. Using all or portion of the nucleic acidsequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:60, SEQ ID NO:62, the cDNA of ATCC 203037 the cDNA ofATCC 203035, the cDNA of ATCC 203036, the cDNA of ATCC PTA-211, the cDNAof ATCC PTA-212, or the cDNA of PTA-213, as a hybridization probe,CARD-3, CARD-4, CARD-5, or CARD-6 nucleic acid molecules can be isolatedusing standard hybridization and cloning techniques (e.g., as describedin Sambrook et al., eds., Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989).

A nucleic acid of the invention can be amplified using cDNA, mRNA orgenomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis. Furthermore, oligonucleotides corresponding toCARD-3, CARD-4, CARD-5, or CARD-6 nucleotide sequences can be preparedby standard synthetic techniques, e.g., using an automated DNAsynthesizer.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQID NO:9, SEQ ID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, the cDNA of ATCC203037, the cDNA of ATCC 203035, or the cDNA of ATCC 203036, or the cDNAof ATCC PTA-211, the cDNA of PTA-212, or the cDNA ofPTA-213, or aportion thereof. A nucleic acid molecule which is complementary to agiven nucleotide sequence is one which is sufficiently complementary tothe given nucleotide sequence that it can hybridize to the givennucleotide sequence thereby forming a stable duplex.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence encoding CARD-3, CARD-4, CARD-5, orCARD-6, for example a fragment which can be used as a probe or primer ora fragment encoding a biologically active portion of CARD-3, CARD-4,CARD-5, or CARD-6. The nucleotide sequence determined from the cloningof the human CARD-3, CARD-4, CARD-5, or CARD-6, and the partial murineCARD-4 gene allows for the generation of probes and primers designed foruse in identifying and/or cloning CARD-3, CARD-4, CARD-5, or CARD-6homologues in other cell types. e.g., from other tissues, as well asCARD-3, CARD-4, CARD-5, or CARD-6 homologues and orthologs from othermammals. The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of thesense or anti-sense sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7,SEQ ID NO:9, SEQ ID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, the cDNA of ATCC203037, the cDNA of ATCC 203035, or the cDNA of ATCC 203036, or the cDNAof ATCC PTA-211, the cDNA of PTA-212, or the cDNA of PTA-213, or of anaturally occurring mutant of one of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:9, SEQ ID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, the cDNA of ATCC203037, the cDNA of ATCC 203035, the cDNA of ATCC 203036, or the cDNA ofATCC PTA-211, the cDNA of PTA-212, or the cDNA of PTA-213.

Probes based on the CARD-3, CARD-4, CARD-5, or CARD-6 nucleotidesequence can be used to detect transcripts or genomic sequences encodingthe same or similar proteins. The probe comprises a label group attachedthereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or anenzyme co-factor. Such probes can be used as a part of a diagnostic testkit for identifying allelic variants and orthologs of the CARD-3,CARD-4, CARD-5, or CARD-6 proteins of the present invention, identifyingcells or tissue which mis-express a CARD-3, CARD-4, CARD-5, or CARD-6protein, such as by measuring a level of a CARD-3, CARD-4, CARD-5, orCARD-6-encoding nucleic acid in a sample of cells from a subject, e.g.,detecting CARD-3, CARD-4, CARD-5, or CARD-6 mRNA levels or determiningwhether a genomic CARD-3, CARD-4, CARD-5, or CARD-6 gene has beenmutated or deleted.

A nucleic acid fragment encoding a “biologically active portion” ofCARD-3, CARD-4, CARD-5, or CARD-6 can be prepared by isolating a portionof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:25, SEQ IDNO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:60, SEQ ID NO:62, the cDNA of ATCC 203037, the cDNA of ATCC 203035,the cDNA of ATCC 203036, or the cDNA of ATCC PTA-211, the cDNA ofPTA-212, or the cDNA of PTA-213, which encodes a polypeptide having aCARD-3, CARD-4, CARD-5, or CARD-6 biological activity, expressing theencoded portion of CARD-3, CARD-4, CARD-5, or CARD-6 protein (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of CARD-3, CARD-4, CARD-5, or CARD-6. For example, anucleic acid fragment encoding a biologically active portion of CARD-3,CARD-4, CARD-5, or CARD-6 includes a CARD domain, e.g., SEQ ID NO:6, SEQID NO:10, SEQ ID NO:28, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, or SEQID NO:62.

The invention further encompasses nucleic acid molecules that differfrom the niucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7,SEQ ID NO:9, SEQ ID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, the cDNA of ATCC203037, the cDNA of ATCC 203035, the cDNA of ATCC 203036, or the cDNA ofATCC PTA-211, the cDNA of PTA-212, or the cDNA of PTA-213, due todegeneracy of the genetic code and thus encode the same CARD-3, CARD-4,CARD-5, or CARD-6 protein as that encoded by the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:25,SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:60, SEQ ID NO:62, the cDNA of ATCC 203037, the cDNA of ATCC203035, the cDNA of ATCC 203036, or the cDNA of ATCC PTA-211, the cDNAof ATCC PTA-212, or the cDNA of ATCC PTA-213.

In addition to the CARD-3, CARD-4, CARD-5, or CARD-6 nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:25,SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:60, SEQ ID NO:62, the cDNA of ATCC 203037, the cDNA of ATCC203035, the cDNA of ATCC 203036, the cDNA of ATCC PTA-211, the cDNA ofATCC PTA-212. or the cDNA of ATCC PTA-213. it will be appreciated bythose skilled in the art that DNA sequence polymorphisms that lead tochanges in the amino acid sequences of CARD-3, CARD-4, CARD-5, or CARD-6may exist within a population (e.g., the human population). Such geneticpolymorphism in the CARD-3, CARD-4, CARD-5, or CARD-6 gene may existamong individuals within a population due to natural allelic variation.As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a CARD-3,CARD-4, CARD-5, or CARD-6 protein, preferably a mammalian CARD-3,CARD-4, CARD-5, or CARD-6 protein. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of theCARD-3, CARD-4, CARD-5, or CARD-6 gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in CARD-3, CARD-4,CARD-5, or CARD-6 that are the result of natural allelic variation andthat do not alter the functional activity of CARD-3, CARD-4, CARD-5, orCARD-6 are intended to be within the scope of the invention. Thus, e.g.,1%, 2%, 3%, 4%, or 5% of the amino acids in CARD-3, CARD-4, CARD-5, orCARD-6 are replaced by another amino acid, preferably the amino acidsare replaced by conservative substitutions.

Moreover, nucleic acid molecules encoding CARD-3, CARD-4, CARD-5, orCARD-6 proteins from other species (CARD-3, CARD-4, CARD-5, or CARD-6orthologs/homologues), which have a nucleotide sequence which differsfrom that of a CARD-3, CARD-4, CARD-5, or CARD-6 disclosed herein, areintended to be within the scope of the invention.

For example, Example 5 describes the murine CARD-4 ortholog and Example14 describes the murine CARD-5 ortholog. Nucleic acid moleculescorresponding to natural allelic variants and homologues of the CARD-3,CARD-4, CARD-5, or CARD-6 cDNA of the invention can be isolated based ontheir similarity to the nucleic acids disclosed herein using the humanor murine cDNAs, or a portion thereof as a hybridization probe accordingto standard hybridization techniques under stringent hybridizationconditions.

In general, an allelic variant of a gene will be readily identifiable asmapping to the same chromosomal location as said gene. For example, inExample 6, the chromosomal location of the human CARD-4 gene isdiscovered to be chromosome 7 close to the SHGC-31928 genetic marker.Allelic variants of human CARD-4 will be readily identifiable as mappingto the human CARD-4 locus on chromosome 7 near genetic markerSHGC-31928.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, 900, 1000, 1300, 1600 or 1931) nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence, preferably the coding sequence, ofSEQ ID NO:1, SEQ ID NO:3, or the cDNA of ATCC 203037. In yet anotherembodiment, an isolated nucleic acid molecule of the invention is atleast 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800,900, 1000, or 1300, 1640, 1900, 2200, 2500, 2800, 3100, or 3382)nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising the nucleotide sequence, preferably thecoding sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:25, SEQ ID NO:27,SEQ ID NO:38, SEQ ID NO:40, the cDNA of ATCC 203035, or the cDNA of ATCC203036. Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention is at least 300 (325, 350, 375, 400, 425, 450,500, 550, 600, 650, 700, 800, 900, 1000, or 1300, 1640, 1900, 2200,2500, 2800, 3100, 3300, 3600, 3900, 4200 or 4209) nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence, preferably the coding sequence, ofSEQ ID NO:42.

In yet another embodiment an isolated nucleic acid molecule of theinvention is at least 300 (350, 400, 450, 500, 550, 600, 650, 700, or740) nucleotides in length and hvbridizes under stringent conditions toa nucleic acid molecule consisting of the nucleotide sequence of SEQ IDNO:48 or SEQ ID NO:50.

In yet another embodiment, an isolated nucleic acid molecule of theinvention is at least 300 (350, 400, 450, 500, 550, 600, 650, 700, or761) nucleotides in length and hybridizes under stringent conditions toa nucleic acid molecule consisting of the nucleotide sequence of SEQ IDNO:60 or SEQ ID NO:62.

In yet another embodiment an isolated nucleic acid molecule of theinvention is at least 300 (350, 400, 450, 500, 550, 600, 650, 700, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5200, or 5252)nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule consisting of the nucleotide sequence of SEQ IDNO:51 or SEQ ID NO:53.

In yet another embodiment, an isolated nucleic acid molecule of theinvention is at least 300 (350, 400, 450, 500, 550, 600, 650, 700, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000) nucleotides in lengthand hybridizes under stringent conditions to a nucleic acid moleculeconsisting of the nucleotide sequence of SEQ ID NO:54 or SEQ ID NO:56.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. An, non-limiting example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at50-65° C. (e.g., 50° C. or 60° C. or 65° C.). Preferably, the isolatednucleic acid molecule of the invention that hybridizes under stringentconditions corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the CARD-3,CARD-4, CARD-5, or CARD-6 sequence that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation into the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:7, SEQ ID NO:9, SEQ ID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, the cDNAof ATCC 203037, the cDNA of ATCC 203035, the cDNA of ATCC 203036, thecDNA of ATCC PTA-211, the cDNA of ATCC PTA-212, or the cDNA of ATCCPTA-213, thereby leading to changes in the amino acid sequence of theencoded protein without altering the functional ability of the protein.For example, one can make nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of CARD-3, CARD-4L/S, CARD-4 splice variant, murine CARD-4protein, human CARD-5 protein, murine CARD-5 protein, or rat CARD-6protein without altering the biological activity, whereas an “essential”amino acid residue is required for biological activity. For example,amino acid residues that are conserved among the CARD-3, CARD-4L/S,CARD-4 splice variant, CARD-4, CARD-5, or CARD-6 proteins of variousspecies are predicted to be particularly unamenable to alteration.

For example, preferred CARD-3, CARD-4, CARD-5, and CARD-6 proteins ofthe present invention, contain at least one CARD domain. Additionally, aCARD-3 protein also contains at least one kinase domain or at least onelinker domain. A CARD domain contains at least one nucleotide bindingdomain or Leucine-rich repeats. Such conserved domains are less likelyto be amenable to mutation. Other amino acid residues, however, (e.g.,those that are not conserved or only semi-conserved among CARD-3,CARD-4, CARD-5, or CARD-6 of various species) may not be essential foractivity and thus are likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding CARD-3, CARD-4, CARD-5, or CARD-6 proteins thatcontain changes in amino acid residues that are not essential foractivity. Such CARD-3, CARD-4, CARD-5, or CARD-6 proteins differ inamino acid sequence from SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:25, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ IDNO:55, or SEQ ID NO:61, and yet retain biological activity. In oneembodiment, the isolated nucleic acid molecule includes a nucleotidesequence encoding a protein that includes an amino acid sequence that isat least about 45% identical, 65%, 75%, 85%, 95%, or 98% identical tothe amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ IDNO:55, or SEQ ID NO:61,

An isolated nucleic acid molecule encoding a CARD-3, CARD-4, CARD-5, orCARD-6 protein having a sequence which differs from that of SEQ ID NO:1,SEQ ID lO NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:25, SEQ ID NO:27, SEQID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:60, SEQ IDNO:62, the cDNA of ATCC 203037, the cDNA of ATCC 203035, the cDNA ofATCC 203036, the cDNA of ATCC PTA-211, the cDNA of ATCC PTA-212, or thecDNA of ATCC PTA-213, can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of CARD-3 (SEQ ID NO:1, SEQ ID NO:3, the cDNA of ATCC 203037)or CARD-4L (SEQ ID NO:7, SEQ ID NO:9, the cDNA of ATCC 203035), orCARD-4S (SEQ ID NO:25, SEQ ID NO:27, the cDNA of ATCC 203036), or humanCARD-4 splice variants (SEQ ID NO:38, SEQ ID NO:40, or murine CARD-4(SEQ ID NO:42), or murine CARD-5 (SEQ ID NO:60, SEQ ID NO:62, the cDNAof PTA-211), or human CARD-5 (SEQ ID NO:48, SEQ ID NO:50, the cDNA ofATCC PTA-213), rat CARD-6 (SEQ ID NO:51, SEQ ID NO:53, the cDNA of ATCCPTA-211), or human CARD-6 (SEQ ID NO:54, SEQ ID NO:56, the cDNA of ATCCPTA-213) such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. Thus, for example, 1%, 2%, 3%, 5%, or 10% of the amino acidscan be replaced by conservative substitution. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in CARD-3, CARD-4, CARD-5, or CARD-6 is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, mutations can be introduced randomly along all orpart of a CARD-3, CARD-4, CARD-5, or CARD-6 coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forCARD-3, CARD-4, CARD-5, or CARD-6 biological activity to identifymutants that retain activity. Following mutagenesis, the encoded proteincan be expressed recombinantly and the activity of the protein can bedetermined.

In an embodiment, a mutant CARD-3, CARD-4, CARD-5, or CARD-6 protein canbe assayed for: (1) the ability to form protein:protein interactionswith proteins in the apoptotic signaling pathway; (2) the ability tobind a CARD-3, CARD-4, CARD-5, or CARD-6 ligand; or (3) the ability tobind to an intracellular target protein. For example, (1) in Example 7,a two-hybrid screening assay for the physical interaction of CARD-3 andCARD-4 is shown, (2) in Example 8, a two-hybrid system assay for theinteraction between CARD-4 and its ligand hNUDC is described, (3) inExample 12, a coimmunoprecipitation assay for the interaction of CARD-3with its ligand CARD-4 is shown, and (4) in Example 17, a two-hybridscreening assay for the physical interaction of CARD-5 and caspase-1,CARD-7, or CARD-5 is shown. In yet another embodiment, a mutant CARD-3,CARD-4, CARD-5, or CARD-6 protein can be assayed for the ability tomodulate cellular proliferation, cellular differentiation, or cellulardeath. For example, in Example 10, assays for the regulation of cellulardeath (apoptosis) by CARD-3 or CARD-4 are described. In yet anotherembodiment, a mutant CARD-3 or CARD-4 protein can be assayed forregulation of a cellular signal transduction pathway. For example, inExamples 9 and 18, an assay for the regulation of the NF-κB pathway byCARD-4 and CARD-5 is described.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire CARD-3, CARD-4,CARD-5, or CARD-6 coding strand, or to only a portion thereof, e.g., allor part of the protein coding region (or open reading frame). Anantisense nucleic acid molecule can be antisense to a noncoding regionof the coding strand of a nucleotide sequence encoding CARD-3, CARD-4,CARD-5, or CARD-6. The noncoding regions (“5′ and 3′ untranslatedregions”) are the 5′ and 3′ sequences which flank the coding region andare not translated into amino acids. Given the coding strand sequencesencoding CARD-3, CARD-4, CARD-5, and CARD-6 disclosed herein, antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick base pairing. The antisense nucleic acid molecule canbe complementary to the entire coding region of CARD-3, CARD-4, CARD-5,or CARD-6L/S mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of CARD-3,CARD-4, CARD-5, or CARD-6 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of CARD-3 mRNA, e.g., an oligonucleotide havingthe sequence CCCTGGTACTTGCCCCTCCGGTAG (SEQ ID NO:34) orCCTGGTACTTGCCCCTCC (SEQ ID NO:35) or of the CARD-4L mRNA, e.g.,TCGTTAAGCCCTTGAAGACAGTG (SEQ ID NO:36) andTCGTTAGCCCTTGAAGACCAGTGAGTGTAG (SEQ ID NO:37) or of the human CARD-5mRNA, e.g., TAGGACCTCGGTACCCGCGCGCGCG (SEQ ID NO:68) or CGCCGGCCCCTAGGACCTCGGTACC (SEQ ID NO:69). An antisense oligonucleotide can be, forexample, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides inlength. An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-aino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described furthr inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a CARD-3,CARD-4, CARD-5, or CARD-6 protein to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule which binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventioninclude direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodieswhich bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisensenucleic acid molecule is placed under the control of a strong pol II orpol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An a-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual B-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330). Theinvention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave CARD-3, CARD-4, CARD-5, or CARD-6 mRNAtranscripts to thereby inhibit translation of CARD-3, CARD-4, CARD-5, orCARD-6 mRNA. A ribozyme having specificity for a CARD-3, CARD-4, CARD-5,or CARD-6-encoding nucleic acid can be designed based upon thenucleotide sequence of a CARD-3, CARD-4, CARD-5, or CARD-6 cDNAdisclosed herein. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved in aCARD-3, CARD-4, CARD-5, or CARD-6-encoding mRNA. See, e.g., Cech et al.U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, CARD-3, CARD-4, CARD-5, or CARD-6 mRNA can be used toselect a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science261:1411-1418.

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, CARD-3, CARD-4, CARD-5, or CARD-6 geneexpression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the CARD-3, CARD-4, CARD-5, orCARD-6 (e.g., the CARD-3, CARD-4, CARD-5, or CARD-6 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the CARD-3, CARD-4, CARD-5, or CARD-6 gene in target cells. Seegenerally, Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992)Ann, N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In embodiments, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1):5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670-675.

PNAs of CARD-3, CARD-4, CARD-5, or CARD-6 can be used for therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, e.g., inducing transcription or translation arrest or inhibitingreplication. PNAs of CARD-3, CARD-4, CARD-5, or CARD-6 can also be used,e.g., in the analysis of single base pair mutations in a gene by, e.g.,PNA directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, e.g., S1 nucleases (Hyrup (1996)supra; or as probes or primers for DNA sequence and hybridization (Hyrup(1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs of CARD-3, CARD-4, CARD-5, or CARD-6 can bemodified, e.g., to enhance their stability or cellular uptake, byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of CARD-3,CARD-4, CARD-5, or CARD-6 can be generated which may combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNAse H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996) supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) supra and Finn et al. (1996) Nucleic Acids Research24(17):3357-63. For example, a DNA chain can be synthesized on a solidsupport using standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a between the PNA and the 5′ end of DNA(Mag et al. (1989) Nucleic Acid Res. 17:5973-88). PNA monomers are thencoupled in a stepwise manner to produce a chimeric molecule with a 5′PNA segment and a 3′ DNA segment (Finn et al. (1996) Nucleic AcidsResearch 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.(1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule. e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

II. Isolated CARD-3, CARD-4, CARD-5, and CARD-6 Proteins andAnti-CARD-3, CARD-4, CARD-5, and CARD-6 Antibodies.

One aspect of the invention pertains to isolated CARD-3, CARD-4, CARD-5,and CARD-6 proteins, and biologically active portions thereof, as wellas polypeptide fragments suitable for use as immunogens to raiseanti-CARD-3, CARD-4, CARD-5, or CARD-6 antibodies. In one embodiment,native CARD-3, CARD-4, CARD-5, or CARD-6 proteins can be isolated fromcells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. In another embodiment CARD-3,CARD-4, CARD-5, or CARD-6 proteins are produced by recombinant DNAtechniques. Alternative to recombinant expression, a CARD-3, CARD-4,CARD-5, or CARD-6 protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theCARD-3, CARD-4, CARD-5, or CARD-6 protein is derived, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized. The language “substantially free of cellular material”includes preparations of CARD-3, CARD-4, CARD-5, or CARD-6 protein inwhich the protein is separated from cellular components of the cellsfrom which it is isolated or recombinantly produced. Thus, CARD-3,CARD-4, CARD-5, or CARD-6 protein that is substantially free of cellularmaterial includes preparations of CARD-3, CARD-4, CARD-5, or CARD-6protein having less than about 30%, 20%, 10%, or 5% (by dry weight) ofnon-CARD-3, CARD-4, CARD-5, or CARD-6 protein (also referred to hereinas a “contaminating protein”). When the CARD-3, CARD-4, CARD-5, orCARD-6 protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When CARD-3, CARD-4, CARD-5, orCARD-6 protein is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. Accordingly such preparationsof CARD-3, CARD-4, CARD-5, or CARD-6 protein have less than about 30%,20%, 10%, 5% (by dry weight) of chemical precursors or non-CARD-3,CARD-4, CARD-5, or CARD-6 chemicals.

Biologically active portions of a CARD-3, CARD-4, CARD-5, or CARD-6protein include peptides comprising amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of the CARD-3,CARD-4, CARD-5, or CARD-6 protein (e.g., the amino acid sequence shownin SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, or SEQ IDNO:61), which include less amino acids than the full length CARD-3,CARD-4, CARD-5, or CARD-6 protein, and exhibit at least one activity ofa CARD-3, CARD-4, CARD-5, or CARD-6 protein. Typically, biologicallyactive portions comprise a domain or motif with at least one activity ofthe CARD-3, CARD-4, CARD-5, or CARD-6 protein. A biologically activeportion of a CARD-3, CARD-4, CARD-5, or CARD-6 protein can be apolypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length. Preferred biologically active polypeptides include one ormore identified CARD-3, CARD-4, CARD-5, or CARD-6 structural domains,e.g., the CARD domain (SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:27, SEQ IDNO:66, SEQ ID NO:67, or SEQ ID NO:68).

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeCARD-3, CARD-4, CARD-5, or CARD-6 protein.

CARD-3, CARD-4, CARD-5, or CARD-6 protein has the amino acid sequenceshown of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, or SEQ IDNO:61. Other useful CARD-3, CARD-4, CARD-5, or CARD-6 proteins aresubstantially identical to SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQID NO:39, SEQ ID NO:41. SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, or SEQID NO:55, or SEQ ID NO:61, and retain the functional activity of theprotein of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, or SEQ IDNO:61, yet differ in amino acid sequence due to natural allelicvariation or mutagenesis. CARD-3 and CARD-4 are involved in activatingcaspases in the apoptotic pathway. For example, in Example 10, CARD-4 isshown to enhance caspase 9 activity. In example 17, CARD-5 is shown tobind to the CARD domain of caspase-1.

A useful CARD-3, CARD-4, CARD-5, or CARD-6 protein is a protein whichincludes an amino acid sequence at least about 45%, preferably 55%, 65%,75%, 85%, 95%, or 99% identical to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, or SEQ ID NO:61, andretains the functional activity of the CARD-3, CARD-4, CARD-5, or CARD-6proteins of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, or SEQ IDNO:61.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions×100).

The determination of percent homology between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Nat'lAcad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program score=100, wordlength=12 to obtainnucleotide sequences similar or homologous to CARD-3, CARD-4, CARD-5, orCARD-6 nucleic acid molecules of the invention. For example, Example 5describes the use of the TBLASTN program to query a database ofsequences of full length and partial cDNA sequences with the humanCARD-4 polypeptide sequence leading to the discovery of murine CARD-4and Example 4 describes the use of BLASTN to query a proprietary ESTdatabase with the 5′ untranslated sequence of CARD-4 leading to thediscovery of two human CARD-4 splice variants. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to CARD-3, CARD-4, CARD-5, orCARD-6 protein molecules of the invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizingBLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, CABIOS (1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

The invention also provides CARD-3, CARD-4, CARD-5, or CARD-6 chimericor fusion proteins. As used herein, a CARD-3, CARD-4, CARD-5, or CARD-6“chimeric protein” or “fusion protein” comprises a CARD-3, CARD-4,CARD-5, or CARD-6 polypeptide operatively linked to a non-CARD-3,CARD-4, CARD-5, or CARD-6 polypeptide. A “CARD-3, CARD-4, CARD-5, orCARD-6 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to all or a portion (preferably a biologicallyactive portion) of a CARD-3, CARD-4, CARD-5, or CARD-6, whereas a“non-CARD-3, CARD-4, CARD-5, or CARD-6 polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially identical to the CARD-3, CARD-4, CARD-5, orCARD-6 protein, e.g., a protein which is different from the CARD-3,CARD-4, CARD-5, or CARD-6 proteins and which is derived from the same ora different organism. Within the fusion protein, the term “operativelylinked” is intended to indicate that the CARD-3, CARD-4, CARD-5, orCARD-6 polypeptide and the non-CARD-3, CARD-4, CARD-5, or CARD-6polypeptide are fused in-frame to each other. The heterologouspolypeptide can be fused to the N-terminus or C-terminus of the CARD-3,CARD-4, CARD-5, or CARD-6 polypeptide.

One useful fusion protein is a GST fusion protein in which the CARD-3,CARD-4, CARD-5, or CARD-6 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant CARD-3, CARD-4, CARD-5, or CARD-6. In another embodiment,the fusion protein contains a signal sequence from another protein. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of CARD-3, CARD-4, CARD-5, or CARD-6 can be increased throughuse of a heterologous signal sequence. For example, the gp67 secretorysequence of the baculovirus envelope protein can be used as aheterologous signal sequence (Current Protocols in Molecular Biology,Ausubel et al., eds., John Wiley & Sons. 1992). Other examples ofeukaryotic heterologous signal sequences include the secretory sequencesof melittin and human placental alkaline phosphatase (Stratagene; LaJolla Calif.). In yet another example, useful prokaryotic heterologoussignal sequences include the phoA secretory signal (Molecular cloning,Sambrook et al, second edition, Cold spring harbor laboratory press,1989) and the protein A secretory signal (Pharmacia Biotech: Piscataway,N.J.).

In yet another embodiment, the fusion protein is a CARD-3, CARD-4,CARD-5, or CARD-6-immunoglobulin fusion protein in which all or part ofCARD-3, CARD-4, CARD-5, or CARD-6 is fused to sequences derived from amember of the immunoglobulin protein family. The CARD-3, CARD-4, CARD-5,or CARD-6-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a CARD-3, CARD-4, CARD-5, orCARD-6 ligand and a CARD-3, CARD-4, CARD-5, or CARD-6 protein on thesurface of a cell, to thereby suppress CARD-3, CARD-4, CARD-5, orCARD-6-mediated signal transduction in vivo. The CARD-3, CARD-4, CARD-5,or CARD-6-immunoglobulin fusion proteins can be used to affect thebioavailability of a CARD-3, CARD-4, CARD-5, or CARD-6 cognate ligand.Inhibition of the CARD-3 ligand/CARD-3, CARD-4 ligand/CARD-4, CARD-5ligand/CARD-5, or CARD-6 ligand/CARD-6 interaction may be usefultherapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g., promoting orinhibiting) cell survival. Moreover, the CARD-3, CARD-4, CARD-5, orCARD-6-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-CARD-3, CARD-4, CARD-5, or CARD-6 antibodiesin a subject, to purify CARD-3, CARD-4, CARD-5, or CARD-6 ligands and inscreening assays to identify molecules which inhibit the interaction ofCARD-3, CARD-4, CARD-5, or CARD-6 with a CARD-3, CARD-4, CARD-5, orCARD-6 ligand.

Preferably, a CARD-3, CARD-4, CARD-5, or CARD-6 chimeric or fusionprotein of the invention is produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques, for example by employing blunt-ended orstagger-ended termini for ligation, restriction enzyme digestion toprovide tor appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Current Protocols in Molecular Biology, Ausubel et al. eds., JohnWiley & Sons: 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide).A CARD-3, CARD-4, CARD-5, or CARD-6-encoding nucleic acid can be clonedinto such an expression vector such that the fusion moietv is linkedin-frame to the CARD-3, CARD-4, CARD-5, or CARD-6 protein.

The present invention also pertains to variants of the CARD-3, CARD-4,CARD-5, or CARD-6 proteins which function as either CARD-3, CARD-4,CARD-5, or CARD-6 agonists (mimetics) or as CARD-3, CARD-4, CARD-5, orCARD-6 antagonists. Variants of the CARD-3, CARD-4, CARD-5, or CARD-6protein can be generated by mutagenesis, e.g., discrete point mutationor truncation of the CARD-3, CARD-4, CARD-5, or CARD-6 protein. Anagonist of the CARD-3, CARD-4, CARD-5, or CARD-6 protein can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the CARD-3, CARD-4, CARD-5, or CARD-6protein. An antagonist of the CARD-3, CARD-4, CARD-5, or CARD-6 proteincan inhibit one or more of the activities of the naturally occurringform of the CARD-3, CARD-4, CARD-5, or CARD-6 protein by, for example,competitively binding to a downstream or upstream member of a cellularsignaling cascade which includes the CARD-3, CARD-4, CARD-5, or CARD-6protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein can have fewer side effects in a subjectrelative to treatment with the naturally occurring form of the CARD-3,CARD-4, CARD-5, or CARD-6 proteins.

Variants of the CARD-3, CARD-4, CARD-5, or CARD-6 protein which functionas either CARD-3, CARD-4, CARD-5, or CARD-6 agonists (mimetics) or asCARD-3, CARD-4, CARD-5, or CARD-6 antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutantsof the CARD-3, CARD-4, CARD-5, or CARD-6 protein for CARD-3, CARD-4,CARD-5, or CARD-6 protein agonist or antagonist activity. In oneembodiment a variegated library of CARD-3, CARD-4, CARD-5, or CARD-6variants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof CARD-3, CARD-4, CARD-5, or CARD-6 variants can be produced by, forexample, enzymatically ligating a mixture of synthetic oligonucleotidesinto gene sequences such that a degenerate set of potential CARD-3,CARD-4, CARD-5, or CARD-6 sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of CARD-3, CARD-4, CARD-5,or CARD-6 sequences therein. There are a variety of methods which can beused to produce libraries of potential CARD-3, CARD-4, CARD-5, or CARD-6variants from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be performed in an automatic DNAsynthesizer and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision. in one mixture of all of the sequences encoding the desiredset of potential CARD-3, CARD-4, CARD-5, or CARD-6 sequences. Methodsfor synthesizing degenerate oligonucleotides are known in the art (see,e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477).

Useful fragments of CARD-3, CARD-4, CARD-5, and CARD-6, includefragments comprising or consisting of a domain or subdomain describedherein. e.g., a kinase domain or a CARD domain.

In addition, libraries of fragments of the CARD-3, CARD-4, CARD-5, orCARD-6 protein coding sequence can be used to generate a variegatedpopulation of CARD-3, CARD-4, CARD-5, or CARD-6 fragments for screeningand subsequent selection of variants of a CARD-3, CARD-4, CARD-5, orCARD-6 protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofa CARD-3, CARD-4, CARD-5, or CARD-6 coding sequence with a nucleaseunder conditions wvherein nicking occurs only about once per moleculedenaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the CARD-3, CARD-4, CARD-5, or CARD-6protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of CARD-3, CARD-4, CARD-5, orCARD-6 proteins. The most widely used techniques, which are amenable tohigh through-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify CARD-3, CARD-4, CARD-5, or CARD-6 variants(Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

An isolated CARD-3, CARD-4, CARD-5, or CARD-6 protein, or a portion orfragment thereof, can be used as an immunogen to generate antibodiesthat bind CARD-3, CARD-4, CARD-5, or CARD-6 using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-lengthCARD-3, CARD-4, CARD-5, or CARD-6 protein can be used or, alternatively,the invention provides antigenic peptide fragments of CARD-3, CARD-4,CARD-5, or CARD-6 for use as immunogens. The antigenic peptide ofCARD-3, CARD-4, CARD-5, or CARD-6 comprises at least 8 (preferably 10,15, 20, or 30) amino acid residues ofthe amino acid sequence shown inSEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:39, SEQ ID NO:41, SEQID NO:43, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, or SEQ ID NO:61 orpolypeptides including amino acids 128-139 or 287-298 of human CARD-4Land encompasses an epitope of CARD-3, CARD-4, CARD-5, or CARD-6 suchthat an antibody raised against the peptide forms a specific immunecomplex with CARD-3, CARD-4, CARD-5, or CARD-6.

Useful antibodies include antibodies which bind to a domain or subdomainof CARD-3, CARD-4, CARD-5, or CARD-6 described herein (e.g., a kinasedomain, a CARD domain, or a leucine-rich domain).

Preferred epitopes encompassed by the antigenic peptide are regions ofCARD-3, CARD-4, CARD-5, or CARD-6 that are located on the surface of theprotein, e.g., hydrophilic regions. Other important criteria include apreference for a terminal sequence, high antigenic index (e.g., aspredicted by Jameson-Wolf algorithm), ease of peptide synthesis (e.g.,avoidance of prolines); and high surface probability (e.g., as predictedby the Emini algorithm; FIG. 8 and FIG. 9).

A CARD-3, CARD-4, CARD-5, or CARD-6 immunogen typically is used toprepare antibodies by immunizing a suitable subject, (e.g., rabbit,goat, mouse or other mammal) with the immunogen. An appropriateimmunogenic preparation can contain, for example recombinantly expressedCARD-3, CARD-4, CARD-5, or CARD-6 protein or a chemically synthesizedCARD-3, CARD-4, CARD-5, or CARD-6 polypeptide. The preparation canfurther include an adjuvant, such as Freund's complete or incompleteadjuvant or similar immunostimulatory agent. Immunization of a suitablesubject with an immunogenic CARD-3, CARD-4, CARD-5, or CARD-6preparation induces a polyclonal anti-CARD-3, CARD-4, CARD-5, or CARD-6antibody response. For example polypeptides including amino acids128-139 or 287-298 of human CARD-4L wvere conjugated to KLH and theresulting conjugates were used to immunize rabbits and polyclonalantibodies that specifically recognize the two immunogen peptides weregenerated.

Accordingly, another aspect of the invention pertains to anti-CARD-3,CARD-4, CARD-5, or CARD-6 antibodies. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen, such as CARD-3,CARD-4, CARD-5, or CARD-6. A molecule which specifically binds toCARD-3, CARD-4, CARD-5, or CARD-6 is a molecule which binds CARD-3,CARD-4, CARD-5, or CARD-6, but does not substantially bind othermolecules in a sample, e.g., a biological sample, which naturallycontains CARD-3, CARD-4, CARD-5, or CARD-6. Examples of immunologicallyactive portions of immunoglobulin molecules include F(ab) and F(ab′)2fragments which can be generated by treating the antibody with an enzymesuch as pepsin. The invention provides polyclonal and monoclonalantibodies that bind CARD-3, CARD-4, CARD-5, or CARD-6. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of CARD-3, CARD-4, CARD-5, or CARD-6. A monoclonalantibody composition thus typically displays a single binding affinityfor a particular CARD-3, CARD-4, CARD-5, or CARD-6 protein with which itimmunoreacts.

Polyclonal anti-CARD-3, CARD-4, CARD-5, or CARD-6 antibodies can beprepared as described above by immunizing a suitable subject with aCARD-3, CARD-4, CARD-5, or CARD-6 immunogen. The anti-CARD-3, CARD-4,CARD-5, or CARD-6 antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized CARD-3, CARD-4,CARD-5, or CARD-6. If desired, the antibody molecules directed againstCARD-3, CARD-4, CARD-5, or CARD-6 can be isolated from the mammal (e.g.,from the blood) and further purified by well-known techniques, such asprotein A chromatography to obtain the IgG fraction. At an appropriatetime after immunization, e.g., when the anti-CARD-3, CARD-4, CARD-5, orCARD-6 antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497, the human Bcell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy. Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing various antibodies monoclonal antibodyhybridomas is well known (see generally Current Protocols in Immunology(1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.).Briefly, an immortal cell line (typically a myeloma) is fused tolymphocytes (typically splenocytes) from a mammal immunized with aCARD-3, CARD-4, CARD-5, or CARD-6 immunogen as described above, and theculture supernatants of the resulting hybridoma cells are screened toidentify a hybridoma producing a monoclonal antibody that binds CARD-3,CARD-4, CARD-5, or CARD-6.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-CARD-3, CARD-4, CARD-5, or CARD-6 monoclonal antibody (see, e.g.,Current Protocols in Immunology, supra; Galfre et al. (1977) Nature266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); andLemer (1981) Yale J. Biol. Med., 54:387-402). Moreover, the ordinarilyskilled worker will appreciate that there are many variations of suchmethods which also would be useful. Typically, the immortal cell line(e.g., a myeloma cell line) is derived from the same mammalian speciesas the lymphocytes. For example, murine hybridomas can be made by fusinglymphocytes from a mouse immunized with an immunogenic preparation ofthe present invention with an immortalized mouse cell line, e.g. amyeloma cell line that is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindCARD-3, CARD-4, CARD-5, or CARD-6, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-CARD-3, CARD-4, CARD-5, or CARD-6 antibody can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withCARD-3, CARD-4, CARD-5, or CARD-6 to thereby isolate immunoglobulinlibrary members that bind CARD-3, CARD-4, CARD-5, or CARD-6. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System.Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit.Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734.

Additionally, recombinant anti-CARD-3, CARD-4, CARD-5, or CARD-6antibodies, such as chimeric and humanized monoclonal antibodies,comprising both human and non-human portions, which can be made usingstandard recombinant DNA techniques, are within the scope of theinvention. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in PCT Publication No. WO 87/02671; EuropeanPatent Application 184,187; European Patent Application 171,496;European Patent Application 173,494; PCT Publication No. WO 86/01533;U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043: Liu et al. (1987) Proc. Natl. Acad.Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sunet al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449;and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison,(1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214;U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060.

An anti-CARD-3, CARD-4, CARD-5, or CARD-6 antibody (e.g., monoclonalantibody) can be used to isolate CARD-3, CARD-4, CARD-5, or CARD-6 bystandard techniques, such as affinity chromatography orimmunoprecipitation. An anti-CARD-3, CARD-4, CARD-5, or CARD-6 antibodycan facilitate the purification of natural CARD-3, CARD-4, CARD-5, orCARD-6 from cells and of recombinantly produced CARD-3, CARD-4, CARD-5,or CARD-6 expressed in host cells. Moreover, an anti-CARD-3, CARD-4,CARD-5, or CARD-6 antibody can be used to detect CARD-3, CARD-4, CARD-5,or CARD-6 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of the CARD-3,CARD-4, CARD-5, or CARD-6 protein. Anti-CARD-3, CARD-4, CARD-5, orCARD-6 antibodies can be used diagnostically to monitor protein levelsin tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, B3-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinvlamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors containing a nucleic acid encoding CARD-3, CARD-4,CARD-5, or CARD-6 (or a portion thereof). As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,expression vectors, are capable of directing the expression of genes towhich they are operatively linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors). However, the invention is intended to include such otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., CARD-3, CARD-4, CARD-5, orCARD-6 proteins mutant forms of CARD-3, CARD-4, CARD-5, or CARD-6 fusionproteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of CARD-3, CARD-4, CARD-5, or CARD-6 in prokaryotic oreukaryotic cells, e.g., bacterial cells such as E. coli, insect cells(using baculovirus expression vectors) yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes:1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes and their cognaterecognition sequences. include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185. AcademicPress, San Diego. Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185. Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment. the CARD-3, CARD-4, CARD-5, or CARD-6 expressionvector is a yeast expression vector. Examples of vectors for expressionin yeast S. cerivisae include pYepSec1 (Baldari et al. (1987) EMBO J.6:229-234), pMFa (Kurjan and Herskowitz. (1982) Cell 30:933-943), pJRY88(Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation,San Diego, Calif.), pGBT9 (Clontech, Palo Alto, Calif.), pGAD10(Clontech, Palo Alto, Calif.), pYADE4 and pYGAE2 and pYPGE2 (Brunelliand Pall, (1993) Yeast 9:1299-1308), pYPGE15 (Brunelli and Pall, (1993)Yeast 9:1309-1318), pACTII (Dr. S. E. Elledge, Baylor College ofMedicine), and picZ (InVitrogen Corp, San Diego, Calif.). For example,in Example 7 the expression of a fusion protein comprising amino acids1-145 of human CARD-4L fused to the DNA-binding domain of S. cerevisiaetranscription factor GAL4 from the yeast expression vector pGBT9 isdescribed. In another example, Example 8 describes the expression of afusion protein comprising amino acids 406-953 of human CARD-4L fused tothe DNA-binding domain of S. cerevisiae transcription factor GAL4 fromthe yeast expression vector pGBT9. In yet another example, Example 7describes the expression of a fusion protein comprising CARD-3 fused tothe transcriptional activation domain of S. cerevisiae transcriptionfactor GAL4 from the yeast expression vector pACTII.

Alternatively, CARD-3, CARD-4, CARD-5, or CARD-6 can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840),pCI (Promega), and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal. (supra). For example, Example 9, Example 10, and Example 12 describethe expression of human CARD-4 or fragments thereof. CARD-3, or bothfrom the mammalian expression vector pCI.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter: Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the a-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to CARD-3, CARD-4, CARD-5, or CARD-6 mRNA. Regulatorysequences operatively linked to a nucleic acid cloned in the antisenseorientation can be chosen which direct the continuous expression of theantisense RNA molecule in a variety of cell types, for instance viralpromoters and/or enhancers, or regulatory sequences can be chosen whichdirect constitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub et al.(Reviews -Trends in Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention or isolated nucleic acidmolecule of the invention has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,CARD-3, CARD-4, CARD-5, or CARD-6 protein can be expressed in bacterialcells such as E. coli insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art. For example, in Example 7 aSaccharomyces cerevisiae host cell for recombinant CARD-4 and CARD-3expression is described, and in Examples 9, 10, 12, 17, and 18, a293Thost cell for expression of CARD-4 or CARD-5 or fragments thereof orCARD-3 are described.

Vector DNA or an isolated nucleic acid molecule of the invention can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Insome cases vector DNA is retained by the host cell. In other cases thehost cell does not retain vector DNA and retains only an isolatednucleic acid molecule of the invention carried by the vector. In somecases, and isolated nucleic acid molecule of the invention is used totransform a cell without the use of a vector.

In order to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding CARD-3, CARD-4, CARD-5, or CARD-6 or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell of the invention, such as a prokarvotic or eukaryotic hostcell in culture can be used to produce (i.e., express) a CARD-3, CARD-4,CARD-5, or CARD-6 protein. Accordingly, the invention further providesmethods for producing CARD-3, CARD-4, CARD-5, or CARD-6 protein usingthe host cells of the invention. In one embodiment, the method comprisesculturing the host cell of the invention (into which a recombinantexpression vector or isolated nucleic acid molecule encoding CARD-3,CARD-4, CARD-5, or CARD-6 has been introduced) in a suitable medium suchthat CARD-3, CARD-4, CARD-5, or CARD-6 protein is produced. In anotherembodiment, the method further comprises isolating CARD-3, CARD-4,CARD-5, or CARD-6 from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichCARD-3, CARD-4, CARD-5, or CARD-6-coding sequences have been introduced.Such host cells can then be used to create non-human transgenic animalsin which exogenous CARD-3, CARD-4, CARD-5, or CARD-6 sequences have beenintroduced into their genome or homologous recombinant animals in whichendogenous CARD-3, CARD-4, CARD-5, or CARD-6 sequences have beenaltered. Such animals are useful for studying the function and/oractivity of CARD-3, CARD-4, CARD-5, or CARD-6 and for identifying and/orevaluating modulators of CARD-3, CARD-4, CARD-5, or CARD-6 activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, etc. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, an“homologous recombinant animal” is a non-human animal, preferably amammal, more preferably a mouse, in which an endogenous CARD-3, CARD-4,CARD-5, or CARD-6 gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingCARD-3, CARD-4, CARD-5, or CARD-6-encoding nucleic acid into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, and allowing the oocyte to develop in a pseudopregnant femalefoster animal. The CARD-3, CARD-4, CARD-5, or CARD-6 cDNA sequence,e.g., that of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQID:25, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:60, SEQ ID NO:62, or the cDNA of ATCC 203037, or thecDNA of ATCC 203035, or the cDNA of ATCC 203036, or the cDNA of ATCCPTA-211, the cDNA of ATCC PTA-212, or the cDNA of ATCC PTA-213) can beintroduced as a transgene into the genome of a non-human animal.Alternatively, a nonhuman homolog or ortholog of the human CARD-3,CARD-4, CARD-5, or CARD-6 gene, such as a mouse CARD-3, CARD-4, CARD-5,or CARD-6 gene, can be isolated based on hybridization to the humanCARD-3, CARD-4, CARD-5, or CARD-6 cDNA and used as a transgene. Forexample, the mouse ortholog of CARD-4, FIG. 15 and SEQ ID NO:42 can beused to make a transgenic animal using standard methods. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to theCARD-3, CARD-4, CARD-5, or CARD-6 transgene to direct expression ofCARD-3, CARD-4, CARD-5, or CARD-6 protein to particular cells. Methodsfor generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the CARD-3, CARD-4, CARD-5, orCARD-6 transgene in its genome and/or expression of CARD-3, CARD-4,CARD-5, or CARD-6 mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodingCARD-3, CARD-4, CARD-5, or CARD-6 can further be bred to othertransgenic animals carrying other transgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a CARD-3, CARD-4, CARD-5, or CARD-6 gene(e.g., a human or a non-human homolog of the CARD-3, CARD-4, CARD-5, orCARD-6 gene, e.g., a murine CARD-3, CARD-4, CARD-5, or CARD-6 gene) intowhich a deletion, addition or substitution has been introduced tothereby alter, e.g., functionally disrupt, the CARD-3, CARD-4, CARD-5,or CARD-6 gene. In an embodiment, the vector is designed such that, uponhomologous recombination, the endogenous CARD-3, CARD-4, CARD-5, orCARD-6 gene is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a “knock out” vector).Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous CARD-3, CARD-4, CARD-5, or CARD-6 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous CARD-3, CARD-4, CARD-5, or CARD-6 protein).In the homologous recombination vector, the altered portion of theCARD-3, CARD-4, CARD-5, or CARD-6 gene is flanked at its 5′ and 3′ endsby additional nucleic acid of the CARD-3, CARD-4, CARD-5, or CARD-6 geneto allow for homologous recombination to occur between the exogenousCARD-3, CARD-4, CARD-5, or CARD-6 gene carried by the vector and anendogenous CARD-3, CARD-4, CARD-5, or CARD-6 gene in an embryonic stemcell. The additional flanking CARD-3, CARD4, CARD-5, or CARD-6 nucleicacid is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several kilobases of flanking DNA(both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedCARD-3, CARD-4, CARD-5, or CARD-6 gene has homologously recombined withthe endogenous CARD-3, CARD-4, CARD-5, or CARD-6 gene are selected (see,e.g., Li et al. (1992) Cell 69:915). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see, e.g., Bradley in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford,1987) pp. 113-152). A chimeric embryo can then be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Bio/Technology 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.In brief, a cell, e.g., a somatic cell, from the transgenic animal canbe isolated and induced to exit the growth cycle and enter Go phase. Thequiescent cell can then be fused, e.g., through the use of electricalpulses, to an enucleated oocyte from an animal of the same species fromwhich the quiescent cell is isolated. The reconstructed oocyte is thencultured such that it develops to morula or blastocyte and thentransferred to pseudopregnant female foster animal. The offspring borneof this female foster animal will be a clone of the animal from whichthe cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The CARD-3, CARD-4, CARD-5, or CARD-6 nucleic acid molecules, CARD-3,CARD-4, CARD-5, or CARD-6 proteins, and anti-CARD-3, CARD-4, CARD-5, orCARD-6 antibodies (also referred to herein as “active compounds”) of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent which modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more addtionalactive compounds.

The agent which modulates expressioon or activity may, for example, be asmall molecule. For example, such small molecules include peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight les thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention. Exemplary doses include milligram or microgram amountsof the small molecule per kilogram of subject or sample weight (e.g.,about 1 microgram per kilogram to about 500 milligrams per kilogram,about 100 micrograms per kilogram to about 5 milligrams per kilogram, orabout 1 microgram per kilogram to about 50 micrograms per kilogram. Itis furthermore understood that appropriate doses of a small moleculedepend upon the potency of the small molecule with respect to theexpression or activity to modulated. Such appropriate doses may bedetermined using the assays described herein. When one or more of thesesmall moleucles is to be administerd to an animal (e.g., a human) inorder to modulate expression or activity of a polypeptide or nucleicacid of the invention, a physician, veterinarian, or researcher may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL? (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a CARD-3, CARD-4, CARD-5, or CARD-6 protein oranti-CARD-3, CARD-4, CARD-5, or CARD-6 antibody) in the required amountin an appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology), c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). A CARD-3, CARD-4, CARD-5, or CARD-6 protein interactswith other cellular proteins and can thus be used for (i) regulation ofcellular proliferation; (ii) regulation of cellular differentiation; and(iii) regulation of cell survival. The isolated nucleic acid moleculesof the invention can be used to express CARD-3, CARD-4, CARD-5, orCARD-6 protein (e.g., via a recombinant expression vector in a host cellin gene therapy applications), to detect CARD-3, CARD-4, CARD-5, orCARD-6 mRNA (e.g., in a biological sample) or a genetic lesion in aCARD-3, CARD-4, CARD-5, or CARD-6 gene, and to modulate CARD-3, CARD-4,CARD-5, or CARD-6 activity. In addition, the CARD-3, CARD-4, CARD-5, orCARD-6 proteins can be used to screen drugs or compounds which modulatethe CARD-3, CARD-4, CARD-5, or CARD-6 activity or expression as well asto treat disorders characterized by insufficient or excessive productionof CARD-3, CARD-4, CARD-5, or CARD-6 protein or production of CARD-3,CARD-4, CARD-5, or CARD-6 protein forms which have decreased or aberrantactivity compared to CARD-3, CARD-4, CARD-5, or CARD-6 wild typeprotein. In addition, the anti-CARD-3, CARD-4, CARD-5, or CARD-6antibodies of the invention can be used to detect and isolate CARD-3,CARD-4, CARD-5, or CARD-6 proteins and modulate CARD-3, CARD-4, CARD-5,or CARD-6 activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides peptidomimetics, small molecules or other drugs)which bind to CARD-3, CARD-4, CARD-5, or CARD-6 proteins or biologicallyactive portions thereof or have a stimulatory or inhibitory effect on,for example, CARD-3, CARD-4, CARD-5, or CARD-6 expression or CARD-3,CARD-4, CARD-5, or CARD-6 activity. An example of a biologically activeportion of human CARD-4 is amino acids 1-145 encoding the CARD domainwhich is sufficient to exhibit CARD-3-binding activity as described inExample 7. Amino acids 406-953 of human CARD-4L comprising the leucinerich repeat domain represent a biologically active portion of CARD-4Lbecause they possess hNUDC-binding activity as described in Example 8.An example of a biologically active portion of human CARD-5 is aminoacids 111-881 (SEQ ID NO:58) encoding the CARD domain.

Among the screening assays provided by the invention are screening toidentify molecules that prevent the dimerization of a CARD-containingpolypeptide of the invention screening to identify molecules which blockthe binding of a CARD containing polypeptide to a CARD-containingpolypeptide of the invention (e.g., CARD-4), screening to identify acompetitive inhibitor of the binding of a nucleotide to the nucleotidebinding site of a CARD-containing polypeptide of the invention, e.g.,human CARD-4L, screening to identify compounds which block theinteraction between the leucine-rich repeat of a CARD-containingpolypeptide of the invention and a ligand which binds to theleucine-rich repeat.

For CARD-6, screening assays can be used to identify molecules whichmodulate a CARD-6 mediated increase in transcription of genes having anAP-1 or NF-κB binding site. For example, expression of a reporter underthe control of NF-κB (or AP-1) is measured in the presence and absenceof a candidate molecule and in the presence and absence of CARD-6 toidentify those molecules which alter expression of the reporter in aCARD-6 dependent manner. In addition, screening assays can be used toidentify molecules which modulate a CARD-6 mediated increase in CHOPphosphorylation. For example, the expression of a reporter gene underthe control of CHOP is measured in the presence and absence of acandidate small molecule and in the presence and absence of CARD-6 toidentify those molecules which alter expression of the reporter in aCARD-6 dependent manner. A screening assay can be carried out toidentify molecules which modulate the CARD-6 mediated increase in CHOPphosphorylation. For example, CHOP phosphorylation is measured in thepresence and absence of a candidate molecule and in the presence andabsence of CARD-6. Phosphorylation of CHOP can be measured using anantibody which binds to phosphorylated CHOP, but not tonon-phosphorylated CHOP.

For nucleotide binding site-containing polypeptides of the invention,e.g., human CARD-4L, screening assays can be used to identify moleculesthat modulate the activity of the nucleotide binding site. For example,molecules can be tested for their ability to modulate, e.g., antagonize,the hydrolysis of ATP by the nucleotide binding site of a polypeptide ofthe invention. Methods of detecting the hydrolysis of ATP by anucleotide binding site are described in, for example, Gadsby et al.,Physiol. Rev. 79:S77-S107, 1999.

For CARD-5, screening assays can be used to identify molecules thatinterfere with the interaction between the CARD domain of CARD-5 and aCARD-5 ligand, e.g., caspase-1, CARD-7, or CARD-5. Additionally,screening assays can be used to identify molecules that modulate aCARD-5-mediated increase in transcription of genes having an NF-κBbinding site.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a CARD-3,CARD-4, CARD-5, or CARD-6 proteins or polypeptides or biologicallyactive portions thereof. The test compounds of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries:spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution: the “one-beadone-compound” library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam (1997) Anticancer Drug Des. 12:145). Examples ofmethods for the synthesis of molecular libraries can be found in theart, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A.90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).

Determining the ability of the test compound to modulate the activity ofCARD-3, CARD-4, CARD-5, or CARD-6 or a biologically active portionthereof can be accomplished, for example, by determining the ability ofthe CARD-3, CARD-4, CARD-5 or CARD-6 protein to bind to or interact witha CARD-3, CARD-4, CARD-5, or CARD-6 target molecule. As used herein, a“target molecule” is a molecule with which a CARD-3, CARD-4, CARD-5, orCARD-6 protein binds or interacts in nature, for example, a moleculeassociated with the internal surface of a cell membrane or a cytoplasmicmolecule. A CARD-3, CARD-4, CARD-5, or CARD-6 target molecule can be anon-CARD-3, CARD-4, CARD-5, or CARD-6 molecule or a CARD-3, CARD-4,CARD-5, or CARD-6 protein or polypeptide of the present invention. Inone embodiment, a CARD-3, CARD-4, CARD-5, or CARD-6 target molecule is acomponent of an apoptotic signal transduction pathway, e.g., CARD-3 andCARD-4. The target, for example, can be a second intracellular proteinwhich has catalytic activity or a protein which facilitates theassociation of downstream signaling molecules with CARD-3, CARD-4,CARD-5, or CARD-6. In another embodiment, CARD-3, CARD-4, CARD-5, orCARD-6 target molecules include CARD-3 because CARD-3 was found to bindto CARD-4 (Examples 7 and 12), hNUDC because hNUDC was found to bind toCARD-4 (Example 8), and caspase-1, CARD-7, and CARD-5 because these wereeach found to bind to CARD-5 (Example 17).

Determining the ability of the test compound to modulate the activity ofCARD-3, CARD-4, CARD-5, or CARD-6 or a biologically active portionthereof can be accomplished, for example, by determining the ability ofthe CARD-3, CARD-4, CARD-5. or CARD-6 protein to bind to or interactwith any of the specific proteins listed in the previous paragraph asCARD-3, CARD-4, CARD-5, or CARD-6 target molecules. In anotherembodiment, CARD-3, CARD-4, CARD-5, or CARD-6 target molecules includeall proteins that bind to a CARD-3, CARD-4, CARD-5, or CARD-6 protein ora fragment thereof in a two-hybrid system binding assay which can beused without undue experimentation to isolate such proteins from cDNA orgenomic two-hybrid system libraries. For example, Example 7 describesthe use of the CARD-4 CARD domain region to identify CARD-3 in atwo-hybrid screen, Example 8 describes the use of the CARD-4 leucinerich repeat domain region to identify hNUDC in a two-hybrid screen, andExample 17 describes the use of the CARD-5 CARD domain region toidentify caspase-1. CARD-7, and CARD-5 in a two-hybrid screen. Thebinding assays described in this section can be cell-based or cell free(described subsequently).

Determining the ability of the CARD-3, CARD-4, CARD-5, or CARD-6 proteinto bind to or interact with a CARD-3, CARD-4, CARD-5, or CARD-6 targetmolecule can be accomplished by one of the methods described above fordetermining direct binding. In an embodiment, determining the ability ofthe CARD-3, CARD-4, CARD-5, or CARD-6 protein to bind to or interactwith a CARD-3, CARD-4, CARD-5, or CARD-6 target molecule can beaccomplished by determining the activity of the target molecule. Forexample, the activity of the target molecule can be determined bydetecting induction of a cellular second messenger of the target (e.g.,intracellular Ca2+, diacylglycerol. IP3, etc.), detectingcatalytic/enzymatic activity of the target on an appropriate substrate,detecting the induction of a reporter gene (e.g., a CARD-3, CARD-4.CARD-5, or CARD-6-responsive regulatory element operatively linked to anucleic acid encoding a detectable marker, e.g. luciferase), ordetecting a cellular response, for example, cell survival, cellulardifferentiation, or cell proliferation. For example, in Example 12CARD-4 is shown to bind to CARD-3 and in Example 10, by monitoring acellular response, CARD-4 is shown to enhance caspase 9 activity, celldeath or apoptosis. Because CARD-3 and CARD-4 enhance caspase 9activity, activity can be monitored by assaying the caspase 9-mediatedapoptosis cellular response or caspase 9 enzymatic activity. Inaddition, and in another embodiment, genes induced by CARD-3, CARD-4,CARD-5, or CARD-6 expression can be identified by expressing CARD-3,CARD-4, CARD-5, or CARD-6 in a cell line and conducting atranscriptional profiling experiment wherein the mRNA expressionpatterns of the cell line transformed with an empty expression vectorand the cell line transformed with a CARD-3, CARD-4, CARD-5, or CARD-6expression vector are compared. The promoters of genes induced byCARD-3, CARD-4, CARD-5, or CARD-6 expression can be operatively linkedto reporter genes suitable for screening such as luciferase, secretedalkaline phosphatase, or beta-galactosidase and the resulting constructscould be introduced into appropriate expression vectors. A recombinantcell line containing CARD-3, CARD-4, CARD-5, or CARD-6 and transfectedwith an expression vector containing a CARD-3, CARD-4, CARD-5, or CARD-6responsive promoter operatively linked to a reporter gene can be used toidentify test compounds that modulate CARD-3, CARD-4, CARD-5, or CARD-6activity by assaying the expression of the reporter gene in response tocontacting the recombinant cell line with test compounds. CARD-3,CARD-4, CARD-5, or CARD-6 agonists can be identified as increasing theexpression of the reporter gene and CARD-3, CARD-4, CARD-5, or CARD-6antagonists can be identified as decreasing the expression of thereporter gene.

In another embodiment of the invention, the ability of a test compoundto modulate the activity of CARD-3, CARD-4, CARD-5, CARD-6 orbiologically active portions thereof can be determined by assaying theability of the test compound to modulate CARD-3, CARD-4, CARD-5, orCARD-6-dependent pathways or processes where the CARD-3, CARD-4, CARD-5,or CARD-6 target proteins that mediate the CARD-3, CARD-4, CARD-5, orCARD-6 effect are known or unknown. Potential CARD-3, CARD-4, CARD-5, orCARD-6-dependent pathways or processes include, but are not limited to,the modulation of cellular signal transduction pathways and theirrelated second messenger molecules (e.g., intracellular Ca²⁺,diacylglycerol, IP3, cAMP etc.), cellular enzymatic activities, cellularresponses (e.g., cell survival, cellular differentiation, or cellproliferation), or the induction or repression of cellular orheterologous mRNAs or proteins. CARD-3, CARD-4, CARD-5, orCARD-6-dependent pathways or processes could be assayed by standardcell-based or cell free assays appropriate for the specific pathway orprocess under study. For example, Examples 9 and 18 describe howexpression of CARD-4S, CARD-4L, or CARD-5 in 293T cells induces theNF-κB pathway as determined by the measurement of a cotransfected NF-κBpathway luciferase reporter gene. In another embodiment, cellscotransfected with CARD-4 or CARD-5 and the NF-κB luciferase reportergene could be contacted with a test compound and test compounds thatblock CARD-4 or CARD-5 activity could be identified by their reductionof CARD-4 or CARD-5-dependent NF-κB pathway luciferase reporter geneexpression. Test compounds that agonize CARD-4 or CARD-5 would beexpected to increase reporter gene expression. In another embodiment,CARD-4 or CARD-5 could be expressed in a cell line and the recombinantCARD-4 or CARD-5-expressing cell line could be contacted with a testcompound. Test compounds that inhibit CARD-4 or CARD-5 activity could beidentified by their reduction of CARD-4 or CARD-5-dependent NF-κBpathway stimulation as measured by the assay of a NF-κB pathway reportergene. NF-κB nuclear localization, IκB phosphorylation or proteolysis, orother standard assays for NF-κB pathway activation known to thoseskilled in the art.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a CARD-3, CARD-4, CARD-5, orCARD-6 protein or biologically active portion thereof with a testcompound and determining the ability of the test compound to bind to theCARD-3, CARD-4, CARD-5, or CARD-6 protein or biologically active portionthereof. Binding of the test compound to the CARD-3, CARD-4, CARD-5, orCARD-6 protein can be determined either directly or indirectly asdescribed above. In one embodiment, a competitive binding assay includescontacting the CARD-3, CARD-4, CARD-5, or CARD-6 protein or biologicallyactive portion thereof with a compound known to bind CARD-3, CARD-4,CARD-5, or CARD-6 to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a CARD-3, CARD-4, CARD-5, or CARD-6 protein, whereindetermining the ability of the test compound to interact with a CARD-3,CARD-4, CARD-5, or CARD-6 protein comprises determining the ability ofthe test compound to preferentially bind to CARD-3, CARD-4, CARD-5, orCARD-6 or biologically active portion thereof as compared to the knownbinding compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting CARD-3, CARD-4, CARD-5, or CARD-6 protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to modulate (e.g., stimulate or inhibit) theactivity of the CARD-3, CARD-4, CARD-5, or CARD-6 protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of CARD-3, CARD-4, CARD-5, or CARD-6can be accomplished, for example, by determining the ability of theCARD-3, CARD-4, CARD-5, or CARD-6 protein to bind to a CARD-3, CARD-4,CARD-5, or CARD-6 target molecule by one of the methods described abovefor determining direct binding. In an alternative embodiment,determining the ability of the test compound to modulate the activity ofCARD-3, CARD-4, CARD-5, or CARD-6 can be accomplished by determining theability of the CARD-3, CARD-4, CARD-5, or CARD-6 protein to furthermodulate a CARD-3, CARD-4, CARD-5, or CARD-6 target molecule. Forexample, the catalytic/enzymatic activity of the target molecule on anappropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theCARD-3, CARD-4, CARD-5, or CARD-6 protein or biologically active portionthereof with a known compound which binds CARD-3, CARD-4, CARD-5, orCARD-6 to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with a CARD-3, CARD-4, CARD-5, or CARD-6 protein, whereindetermining the ability of the test compound to interact with a CARD-3,CARD-4, CARD-5, or CARD-6 protein comprises determining the ability ofthe CARD-3. CARD-4, CARD-5, or CARD-6 protein to preferentially bind toor modulate the activity of a CARD-3, CARD-4, CARD-5, or CARD-6 targetmolecule. The cell-free assays of the present invention are amenable touse of both the soluble form or the membrane-associated form of CARD-3,CARD-4, CARD-5, or CARD-6. A membrane-associated form of CARD-3, CARD-4,CARD-5, or CARD-6 refers to CARD-3, CARD-4, CARD-5, or CARD-6 thatinteracts with a membrane-bound target molecule. In the case ofcell-free assays comprising the membrane-associated form of CARD-3,CARD-4, CARD-5, or CARD-6, it may be desirable to utilize a solubilizingagent such that the membrane-associated form of CARD-3, CARD-4, CARD-5,or CARD-6 is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either CARD-3, CARD-4,CARD-5, or CARD-6 or its target molecule to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound toCARD-3, CARD-4, CARD-5, or CARD-6, or interaction of CARD-3, CARD-4,CARD-5, or CARD-6 with a target molecule in the presence and absence ofa candidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/CARD-3, CARD-4, CARD-5, or CARD-6 fusionproteins or glutathione-S-transferase/target fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or CARD-3, CARD-4, CARD-5, or CARD-6protein, and the mixture incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtitre plate wells are washed to remove anyunbound components, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of CARD-3, CARD-4, CARD-5, or CARD-6 binding or activitydetermined using standard techniques. In an alternative embodiment, MYCor HA epitope tag CARD-3 or CARD-4 fusion proteins or MYC or HA epitopetag target fusion proteins can be adsorbed onto anti-MYC or anti-HAantibody coated microbeads or onto anti-MYC or anti-HA antibody coatedmicrotitre plates, which are then combined with the test compound or thetest compound and either the non-adsorbed target protein or CARD-3 orCARD-4 protein, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads or microtitre plate wells are washed toremove any unbound components, the matrix immobilized in the case ofbeads, complex determined either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of CARD-3 or CARD-4 binding or activitydetermined using standard techniques. Example 12 describes an HA epitopetagged CARD-4 protein that physically interacts in acoimmunoprecipitation assay with MYC epitope tagged CARD-3, In anembodiment of the invention, HA epitope tagged CARD-4 could be used incombination with MYC epitope CARD-3 in the sort of protein-proteininteraction assay described earlier in this paragraph.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, CARD-3, CARD-4,CARD-5, or CARD-6 or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated CARD-3 or CARD-4 ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals. Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with CARD-3, CARD-4, CARD-5, CARD-6 or targetmolecules but which do not interfere with binding of the protein to itstarget molecule can be derivatized to the wells of the plate, andunbound target or protein trapped in the wells by antibody conjugation.Methods for detecting such complexes in addition to those describedabove for the GST-immobilized complexes and epitope tag immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the CARD-3, CARD-4, CARD-5, or CARD-6 or target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the CARD-3, CARD-4, CARD-5, CARD-6 or targetmolecule.

In another embodiment, modulators of CARD-3, CARD-4, CARD-5, or CARD-6expression are identified in a method in which a cell is contacted witha candidate compound and the expression of the CARD-3, CARD-4, CARD-5,or CARD-6 promoter. mRNA or protein in the cell is determined. The levelof expression of CARD-3, CARD-4, CARD-5, or CARD-6 mRNA or protein inthe presence of the candidate compound is compared to the level ofexpression of CARD-3, CARD-4, CARD-5, or CARD-6 mRNA or protein in theabsence of the candidate compound. The candidate compound can then beidentified as a modulator of CARD-3, CARD-4, CARD-5, or CARD-6expression based on this comparison. For example, when expression ofCARD-3, CARD-4, CARD-5, or CARD-6 mRNA or protein is greater(statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of CARD-3, CARD-4, CARD-5, or CARD-6 mRNA or proteinexpression. Alternatively, when expression of CARD-3, CARD-4, CARD-5, orCARD-6 mRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of CARD-3, CARD-4, CARD-5, orCARD-6 mRNA or protein expression. The level of CARD-3, CARD-4, CARD-5,or CARD-6 mRNA or protein expression in the cells can be determined bymethods described herein for detecting CARD-3, CARD-4, CARD-5, or CARD-6mRNA or protein. The activity of the CARD-3, CARD-4, CARD-5, or CARD-6promoter can be assayed by linking the CARD-3, CARD-4, CARD-5, or CARD-6promoter to a reporter gene such as luciferase, secreted alkalinephosphatase, or beta-galactosidase and introducing the resultingconstruct into an appropriate vector, transfecting a host cell line, andmeasuring the activity of the reporter gene in response to testcompounds. For example, two CARD-4-specific mRNAs were detected in aNorthern blotting experiment, one of 4.6 kilobases and the other of6.5-7.0 kilobases (Example 11). In Example 11. CARD-4-specific mRNAspecies were found to be widely distributed in the tissues and celllines studied.

In yet another aspect of the invention, the CARD-3, CARD-4, CARD-5, orCARD-6 proteins can be used as “bait proteins” in a two-hybrid assay orthree hybrid assay (see. e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO94/10300), to identify other proteins, which bind to or interact withCARD-3, CARD-4, CARD-5, or CARD-6 (“CARD-3, CARD-4, CARD-5, orCARD-6-binding proteins” or “CARD-3, CARD-4, CARD-5, or CARD-6-bp”) andmodulate CARD-3, CARD-4, CARD-5, or CARD-6 activity. Such CARD-3,CARD-4, CARD-5, or CARD-6-binding proteins are also likely to beinvolved in the propagation of signals by the CARD-3, CARD-4, CARD-5, orCARD-6 proteins as, for example, upstream or downstream elements of theCARD-3, CARD-4, CARD-5, or CARD-6 pathway. For example, Example 7describes the construction of a two-hybrid screening bait constructincluding human CARD-4L amino acids 1-145 comprising the CARD domain andthe use of this bait construct to screen human mammary gland andprostate gland two-hybrid libraries resulting in the identification ofhuman CARD-3 as a CARD-4 interacting protein. In another example,Example 8 describes the construction of a two-hybrid screening baitconstruct including human CARD-4 amino acids 406-953 comprising the LRRdomain and the use of this bait construct to screen a human mammarygland two-hybrid libraries resulting in the identification of hNUDC as aCARD-4 interacting protein. In another example, Example 17 describes theconstruction of a two-hybrid screening bait construct including humanCARD-5 CARD domain and the use of this bait construct to screen a panelof 26 CARD domains, resulting in the identification of caspase-1.CARD-7, and CARD-5 as CARD-5 interacting proteins.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for CARD-3, CARD-4,CARD-5, or CARD-6 is fused to a gene encoding the DNA binding domain ofa known transcription factor (e.g., GAL-4). In the other construct, aDNA sequence, from a library of DNA sequences, that encodes anunidentified protein (“prey” or “sample”) is fused to a gene that codesfor the activation domain of the known transcription factor. If the“bait” and the “prey” proteins are able to interact, in vivo, forming anCARD-3, CARD-4, CARD-5, or CARD-6-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with CARD-3, CARD-4, CARD-5,or CARD-6.

In an embodiment of the invention, the ability of a test compound tomodulate the activity of CARD-3, CARD-4, CARD-5, or CARD-6, or abiologically active portion thereof can be determined by assaying theability of the test compound to block the binding of CARD-3, CARD-4,CARD-5, or CARD-6 to its target proteins in a two-hybrid system assay.Example 7 describes a two-hybrid system assay for the interactionbetween CARD-3 and CARD-4, Example 8 describes a two-hybrid system assayfor the interaction between CARD-4 and its target protein hNUDC, andExample 17 describes a two-hybrid system assay for the interactionbetween CARD-5 and its target proteins caspase-1, CARD-7, and CARD-5. Toscreen for test compounds that block the interaction between CARD-3,CARD-4, CARD-5 and their target proteins, which include but are notlimited to CARD-3, CARD-4, hNUDC, caspase-1, CARD-7, and CARD-5, a yeasttwo-hybrid screening strain coexpressing the interacting bait and preyconstructs, for example, a CARD-4 bait construct and a CARD-3 preyconstruct as described in Example 7, is contacted with the test compoundand the activity of the two-hybrid system reporter gene, usually HIS3,lacZ, or URA3 is assayed. If the strain remains viable but exhibits asignificant decrease in reporter gene activity, this would indicate thatthe test compound has inhibited the interaction between the bait andprey proteins. This assay could be automated for high throughput drugscreening purposes. In another embodiment of the invention, CARD-3,CARD-4, CARD-5, or CARD-6 and their target proteins could be configuredin the reverse two-hybrid system (Vidal et al. (1996) Proc. Natl. Acad.Sci. USA 93:10321-6 and Vidal et al. (1996) Proc. Natl. Acad. Sci. USA93:10315-20) designed specifically for efficient drug screening. In thereverse two-hybrid system, inhibition of a CARD-3 or CARD-4 physicalinteraction with a target protein would result in induction of areporter gene in contrast to the normal two-hybrid system whereinhibition of CARD-3, CARD-4, CARD-5, or CARD-6 physical interactionwith a target protein would lead to reporter gene repression. Thereverse two-hybrid system is preferred for drug screening becausereporter gene induction is more easily assayed than report generepression.

Alternative embodiments of the invention are proteins found tophysically interact with proteins that bind to CARD-3, CARD-4, CARD-5,or CARD-6. CARD-3, CARD-4, CARD-5, or CARD-6 interactors, including butnot limited to hNUDC and CARD-3, could be configured into two-hybridsystem baits and used in two-hybrid screens to identify additionalmembers of the CARD-3, CARD-4, CARD-5, or CARD-6 pathway. Theinteractors of CARD-3, CARD-4, CARD-5, or CARD-6 interactors identifiedin this way could be useful targets for therapeutic intervention inCARD-3, CARD-4, CARD-5, or CARD-6 related diseases and pathologies andan assay of their enzymatic or binding activity could be useful for theidentification of test compounds that modulate CARD-3, CARD-4, CARD-5,or CARD-6 activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, CARD-3, CARD-4, CARD-5, or CARD-6 nucleic acidmolecules described herein or fragments thereof, can be used to map thelocation of CARD-3, CARD-4, CARD-5, or CARD-6 genes on a chromosome. Themapping of the CARD-3, CARD-4, CARD-5, or CARD-6 sequences tochromosomes is an important first step in correlating these sequenceswith genes associated with disease.

Briefly, CARD-3, CARD-4, CARD-5, or CARD-6 genes can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp in length)from the CARD-3, CARD-4, CARD-5, or CARD-6 sequences. Computer analysisof CARD-3, CARD-4, CARD-5, or CARD-6 sequences can be used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the CARD-3, CARD-4, CARD-5, or CARD-6 sequences willyield an amplified fragment. For example, in Example 6, humanCARD-4-specific PCR primers were used to screen DNAs from a somatic cellhybrid panel showing that human CARD-4 maps to chromosome 7 close to theSHGC-31928 genetic marker.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio et al. (1983) Science220:919-924). Somatic cell hybrids containing only fragments of humanchromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the CARD-3,CARD-4, CARD-5, or CARD-6 sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa CARD-3, CARD-4, CARD-5, or CARD-6 sequence to its chromosome includein situ hybridization (described in Fan et al. (1990) Proc. Natl. Acad.Sci. USA 87:6223-27), pre-screening with labeled flow-sortedchromosomes, and pre-selection by hybridization to chromosome specificcDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., (Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press. New York, 1988)).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature,325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the CARD-3, CARD-4,CARD-5, or CARD-6 gene can be determined. If a mutation is observed insome or all of the affected individuals but not in any unaffectedindividuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffectedindividuals generally involves first looking for structural alterationsin the chromosomes such as deletions or translocations that are visiblefrom chromosome spreads or detectable using PCR based on that DNAsequence. Ultimately, complete sequencing of genes from severalindividuals can be performed to confirm the presence of a mutation andto distinguish mutations from polymorphisms.

2. Tissue Typing

The CARD-3, CARD-4, CARD-5, or CARD-6 sequences of the present inventioncan also be used to identify individuals from minute biological samples.The United States military, for example, is considering the use ofrestriction fragment length polymorphism (RFLP) for identification ofits personnel. In this technique, an individual's genomic DNA isdigested with one or more restriction enzymes, and probed on a Southernblot to yield unique bands for identification. This method does notsuffer from the current limitations of “Dog Tags” which can be lost,switched, or stolen, making positive identification difficult. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the CARD-3, CARD-4, CARD-5, or CARD-6 sequences describedherein can be used to prepare two PCR primers from the 5′ and 3′ ends ofthe sequences. These primers can then be used to amplify an individual'sDNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The CARD-3, CARD-4, CARD-5, or CARD-6 sequences of the inventionuniquely represent portions of the human genome. Allelic variationoccurs to some degree in the coding regions of these sequences, and to agreater degree in the noncoding regions. It is estimated that allelicvariation between individual humans occurs with a frequency of aboutonce per each 500 bases. Each of the sequences described herein can, tosome degree, be used as a standard against which DNA from an individualcan be compared for identification purposes. Because greater numbers ofpolymorphisms occur in the noncoding regions, fewer sequences arenecessary to differentiate individuals. The noncoding sequences of SEQID NO:1, SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, and SEQ ID NO:60 cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:50, SEQ ID NO:53, SEQID NO:56, and SEQ ID NO:62 are used, a more appropriate number ofprimers for positive individual identification would be 500-2,000.

If a panel of reagents from CARD-3, CARD-4, CARD-5, or CARD-6 sequencesdescribed herein is used to generate a unique identification databasefor an individual, those same reagents can later be used to identifytissue from that individual. Using the unique identification database,positive identification of the individual, living or dead, can be madefrom extremely small tissue samples.

3. Use of Partial Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids. e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:25,SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:51.SEQ ID NO: 54, and SEQ ID NO:60 are particularly appropriate for thisuse as greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the CARD-3, CARD-4, CARD-5,or CARD-6 sequences or portions thereof. e.g., fragments derived fromthe noncoding regions of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:25, SEQ IDNO:38. SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:51. SEQ IDNO:54, and SEQ ID NO:60 which have a length of at least 20 or 30 bases.

The sequences described herein can further be used to providepolynucleotide reagents. e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue. This can be very useful in caseswhere a forensic pathologist is presented with a tissue of unknownorigin. Panels of such CARD-3, CARD-4, CARD-5, or CARD-6 probes can beused to identify tissue by species and/or by organ type.

In a similar fashion, these reagents. e.g., CARD-3, CARD-4, CARD-5, orCARD-6 primers or probes can be used to screen tissue culture forcontamination (i.e., screen for the presence of a mixture of differenttypes of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningCARD-3, CARD-4, CARD-5, or CARD-6 protein and/or nucleic acid expressionas well as CARD-3, CARD-4, CARD-5, or CARD-6 activity, in the context ofa biological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant CARD-3,CARD-4, CARD-5, or CARD-6 expression or activity. The invention alsoprovides for prognostic (or predictive) assays for determining whetheran individual is at risk of developing a disorder associated withCARD-3, CARD-4, CARD-5, or CARD-6 protein, nucleic acid expression oractivity. For example, mutations in a CARD-3, CARD-4, CARD-5, or CARD-6gene can be assayed in a biological sample. Such assays can be used forprognostic or predictive purpose to thereby prophylactically treat anindividual prior to the onset of a disorder characterized by orassociated with CARD-3, CARD-4, CARD-5, or CARD-6 protein, nucleic acidexpression or activity.

Another aspect of the invention provides methods for determining CARD-3,CARD-4, CARD-5, or CARD-6 protein, nucleic acid expression or CARD-3,CARD-4, CARD-5, or CARD-6 activity in an individual to thereby selectappropriate therapeutic or prophylactic agents for that individual(referred to herein as “pharmacogenomics”). Pharmacogenomics allows forthe selection of agents (e.g., drugs) for therapeutic or prophylactictreatment of an individual based on the genotype of the individual(e.g., the genotype of the individual examined to determine the abilityof the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof CARD-3, CARD-4, CARD-5, or CARD-6 in clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of CARD-3,CARD-4, CARD-5, or CARD-6 in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting CARD-3, CARD-4,CARD-5, or CARD-6 protein or nucleic acid (e.g., mRNA, genomic DNA) thatencodes CARD-3, CARD-4, CARD-5, or CARD-6 protein such that the presenceof CARD-3, CARD-4, CARD-5, or CARD-6 is detected in the biologicalsample. An agent for detecting CARD-3, CARD-4, CARD-5, or CARD-6 mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toCARD-3, CARD-4, CARD-5, or CARD-6 mRNA or genomic DNA. The nucleic acidprobe can be, for example, a full-length CARD-3, CARD-4, CARD-5, orCARD-6 nucleic acid such as the nucleic acid of SEQ ID NO:1 or 3, SEQ IDNO:7 or 9, SEQ ID NO:25 or 27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:48 or 50, SEQ ID NO:51 or SEQ ID NO:53 or SEQ ID NO:54 or SEQID NO:56, SEQ ID NO:60 or 62, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to mRNA or genomic DNA, or a human CARD-4 splice variant suchas the nucleic acid of SEQ ID NO:38 or SEQ ID NO:40. Other suitableprobes for use in the diagnostic assays of the invention are describedherein. For example, Example 11 describes the use of a nucleic acidprobe to detect CARD-4 mRNAs in human tissues and cell lines and theprobe used in this experiment could be used for a diagnostic assay.

An agent for detecting CARD-3, CARD-4, CARD-5, or CARD-6 protein can bean antibody capable of binding to CARD-3, CARD-4, CARD-5, or CARD-6protein., preferably an antibody with a detectable label. Antibodies canbe polyclonal, or more preferably, monoclonal. For example, polypeptidescorresponding to amino acids 128-139 and 287-298 of human CARD-4L wereused to immunize rabbits and produce polyclonal antibodies thatspecifically recognize human CARD-4L. An intact antibody, or a fragmentthereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, withregard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect CARD-3, CARD-4, CARD-5, or CARD-6 mRNA, protein, orgenomic DNA in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of CARD-3, CARD-4, CARD-5, orCARD-6 mRNA include Northern hybridizations and in situ hybridizations.For example, Example 11 contains the use of a human CARD-4L nucleic acidprobe for a Northern blotting analysis of mRNA species encoded by humanCARD-4L detected in RNA samples from human tissues and cell lines. Invitro techniques for detection of CARD-3 or CARD-4 protein includeenzyme linked immunosorbent assays (ELISAs), Western blots.immunoprecipitations and immunofluorescence. In vitro techniques fordetection of CARD-3, CARD-4, CARD-5, or CARD-6 genomic DNA includeSouthern hybridizations. Furthermore, in vivo techniques for detectionof CARD-3, CARD-4, CARD-5, or CARD-6 protein include introducing into asubject a labeled anti-CARD-3, CARD-4, CARD-5, or CARD-6 antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. An biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting CARD-3, CARD-4, CARD-5, orCARD-6 protein, mRNA, or genomic DNA, such that the presence of CARD-3,CARD-4, CARD-5, or CARD-6 protein. mRNA or genomic DNA is detected inthe biological sample, and comparing the presence of CARD-3, CARD-4,CARD-5, or CARD-6 protein, mRNA or genomic DNA in the control samplewith the presence of CARD-3, CARD-4, CARD-5, or CARD-6 protein, mRNA orgenomic DNA in the test sample.

The invention also encompasses kits for detecting the presence ofCARD-3, CARD-4, CARD-5, or CARD-6 in a biological sample (a testsample). Such kits can be used to determine if a subject is sufferingfrom or is at increased risk of developing a disorder associated withaberrant expression of CARD-3, CARD-4, CARD-5, or CARD-6 (e.g., animmunological disorder). For example, the kit can comprise a labeledcompound or agent capable of detecting CARD-3, CARD-4, CARD-5, or CARD-6protein or mRNA in a biological sample and means for determining theamount of CARD-3, CARD-4, CARD-5, or CARD-6 in the sample (e.g., ananti-CARD-3, CARD-4, CARD-5, or CARD-6 antibody or an oligonucleotideprobe which binds to DNA encoding CARD-3, CARD-4, CARD-5, or CARD-6,e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IS NO:48,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:60, or SEQ ID NO:62). Kits may also include instruction forobserving that the tested subject is suffering from or is at risk ofdeveloping a disorder associated with aberrant expression of CARD-3,CARD-4, CARD-5, or CARD-6 if the amount of CARD-3, CARD-4, CARD-5, orCARD-6 protein or mRNA is above or below a normal level.

For antibody-based kits, the kit may comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to CARD-3,CARD-4, CARD-5, or CARD-6 protein; and, optionally, (2) a second,different antibody which binds to CARD-3, CARD-4, CARD-5, or CARD-6protein or the first antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit may comprise, for example: (1) aoligonucleotide. e.g., a detectably labelled oligonucleotide, whichhybridizes to a CARD-3, CARD-4, CARD-5, or CARD-6 nucleic acid sequenceor (2) a pair of primers useful for amplifying a CARD-3, CARD-4, CARD-5,or CARD-6 nucleic acid molecule.

The kit may also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit may also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit may also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of CARD-3, CARD-4, CARD-5, orCARD-6.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant CARD-3,CARD-4, CARD-5, or CARD-6 expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with CARD-3, CARD-4, CARD-5, orCARD-6 protein, nucleic acid expression or activity. Alternatively, theprognostic assays can be utilized to identify a subject having or atrisk for developing such a disease or disorder. Thus, the presentinvention provides a method in which a test sample is obtained from asubject and CARD-3, CARD-4, CARD-5, or CARD-6 protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of CARD-3,CARD-4, CARD-5, or CARD-6 protein or nucleic acid is diagnostic for asubject having or at risk of developing a disease or disorder associatedwith aberrant CARD-3, CARD-4, CARD-5, or CARD-6 expression or activity.As used herein, a “test sample” refers to a biological sample obtainedfrom a subject of interest. For example, a test sample can be abiological fluid (e.g., serum), cell sample, or tissue. Furthermore, theprognostic assays described herein can be used to determine whether asubject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with aberrantCARD-3, CARD-4, CARD-5, or CARD-6 expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with a specific agent or class of agents (e.g.,agents of a type which decrease CARD-3, CARD-4, CARD-5, or CARD-6activity). Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant CARD-3, CARD-4, CARD-5, or CARD-6expression or activity in which a test sample is obtained and CARD-3,CARD-4, CARD-5, or CARD-6 protein or nucleic acid is detected (e.g.,wherein the presence of CARD-3, CARD-4, CARD-5, or CARD-6 protein ornucleic acid is diagnostic for a subject that can be administered theagent to treat a disorder associated with aberrant CARD-3, CARD-4,CARD-5, or CARD-6 expression or activity).

The methods of the invention can also be used to detect genetic lesionsor mutations in a CARD-3, CARD-4, CARD-5, or CARD-6 gene, therebydetermining if a subject with the lesioned gene is at risk for adisorder characterized by aberrant cell proliferation and/ordifferentiation. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion characterized by at least one of analteration affecting the integrity of a gene encoding a CARD-3, CARD-4,CARD-5, or CARD-6-protein, or the mis-expression of the CARD-3, CARD-4,CARD-5, or CARD-6 gene. For example, such genetic lesions can bedetected by ascertaining the existence of at least one of 1) a deletionof one or more nucleotides from a CARD-3, CARD-4, CARD-5, or CARD-6gene; 2) an addition of one or more nucleotides to a CARD-3, CARD-4,CARD-5, or CARD-6 gene; 3) a substitution of one or more nucleotides ofa CARD-3, CARD-4, CARD-5, or CARD-6 gene; 4) a chromosomal rearrangementof a CARD-3, CARD-4, CARD-5, or CARD-6 gene; 5) an alteration in thelevel of a messenger RNA transcript of a CARD-3, CARD-4, CARD-5, orCARD-6 gene; 6) aberrant modification of a CARD-3, CARD-4, CARD-5, orCARD-6 gene, such as of the methylation pattern of the genomic DNA; 7)the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a CARD-3, CARD-4, CARD-5, or CARD-6 gene (e.g, caused by amutation in a splice donor or splice acceptor site); 8) a non-wild typelevel of a CARD-3, CARD-4, CARD-5, or CARD-6-protein; 9) allelic loss ofa CARD-3, CARD-4, CARD-5, or CARD-6 gene; and 10) inappropriatepost-translational modification of a CARD-3, CARD-4, CARD-5, orCARD-6-protein. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in aCARD-3, CARD-4, CARD-5, or CARD-6 gene. A biological sample is aperipheral blood leukocyte sample isolated by conventional means from asubject.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4.683,195 and 4.683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the CARD-3 orCARD-4-gene (see, e.g., Abravaya et al. (1995) Nucleic Acids Res.23:675-682). This method can include the steps of collecting a sample ofcells from a patient, isolating nucleic acid (e.g., genomic, mRNA orboth) from the cells of the sample, contacting the nucleic acid samplewith one or more primers which specifically hybridize to a CARD-3,CARD-4, CARD-5, or CARD-6 gene under conditions such that hybridizationand amplification of the CARD-3, CARD-4, CARD-5, or CARD-6-gene (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a CARD-3, CARD-4, CARD-5, orCARD-6 gene from a sample cell can be identified by alterations inrestriction enzyme cleavage patterns. For example, sample and controlDNA is isolated, amplified (optionally), digested with one or morerestriction endonucleases, and fragment length sizes are determined bygel electrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in CARD-3, CARD-4, CARD-5, orCARD-6 can be identified by hybridizing a sample and control nucleicacids, e.g., DNA or RNA, to high density arrays containing hundreds orthousands of oligonucleotides probes (Cronin et al. (1996) HumanMutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). Forexample, genetic mutations in CARD-3 or CARD-4 can be identified intwo-dimensional arrays containing light-generated DNA probes asdescribed in Cronin et al. supra. Briefly, a first hybridization arrayof probes can be used to scan through long stretches of DNA in a sampleand control to identify base changes between the sequences by makinglinear arrays of sequential overlapping probes. This step allows theidentification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the CARD-3, CARD-4,CARD-5, or CARD-6 gene and detect mutations by comparing the sequence ofthe sample CARD-3, CARD-4, CARD-5, or CARD-6 with the correspondingwild-type (control) sequence. Examples of sequencing reactions includethose based on techniques developed by Maxam and Gilbert ((1977) Proc.Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci.USA 74:5463). It is also contemplated that any of a variety of automatedsequencing procedures can be utilized when performing the diagnosticassays ((1995) Bio/Techniques 19:448), including sequencing by massspectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al.(1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl.Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the CARD-3 or CARD-4 geneinclude methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type CARD-3, CARD-4, CARD-5, orCARD-6 sequence with potentially mutant RNA or DNA obtained from atissue sample. The double-stranded duplexes are treated with an agentwhich cleaves single-stranded regions of the duplex such as which willexist due to basepair mismatches between the control and sample strands.For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNAhybrids treated with S1 nuclease to enzymatically digesting themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,e.g., Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymol. 217:286-295. In an embodiment, the control DNAor RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in CARD-3, CARD-4, CARD-5, orCARD-6 cDNAs obtained from samples of cells. For example, the mutYenzyme of E. Coli cleaves A at G/A mismatches and the thymidine DNAglycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.(1994) Carcinogenesis 15:1657-1662). According to an exemplaryembodiment, a probe based on a CARD-3, CARD-4, CARD-5, or CARD-6sequence, e.g., a wild-type CARD-3, CARD-4, CARD-5, or CARD-6 sequence,is hybridized to a cDNA or other DNA product from a test cell(s). Theduplex is treated with a DNA mismatch repair enzyme, and the cleavageproducts, if any, can be detected from electrophoresis protocols or thelike. See, e.g., U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in CARD-3, CARD-4, CARD-5, or CARD-6 genes.For example, single strand conformation polymorphism (SSCP) may be usedto detect differences in electrophoretic mobility between mutant andwild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA:86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi(1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments ofsample and control CARD-3 or CARD-4 nucleic acids will be denatured andallowed to renature. The secondary structure of single-stranded nucleicacids varies according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In an embodiment, the subject method utilizesheteroduplex analysis to separate double stranded heteroduplex moleculeson the basis of changes in electrophoretic mobility (Keen et al. (1991)Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a CARD-3, CARD-4,CARD-5, or CARD-6 gene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which CARD-3 or CARD-4 is expressed may be utilized inthe prognostic assays described herein.

3. Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onCARD-3, CARD-4, CARD-5, or CARD-6 activity (e.g., CARD-3, CARD-4,CARD-5, or CARD-6 gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) disorders (e.g., an immunologicaldisorder) associated with aberrant CARD-3, CARD-4, CARD-5, or CARD-6activity. In conjunction with such treatment, the pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) of theindividual may be considered. Differences in metabolism of therapeuticscan lead to severe toxicity or therapeutic failure by altering therelation between dose and blood concentration of the pharmacologicallyactive drug. Thus, the pharmacogenomics of the individual permits theselection of effective agents (e.g., drugs) for prophylactic ortherapeutic treatments based on a consideration of the individual'sgenotype. Such pharmacogenomics can further be used to determineappropriate dosages and therapeutic regimens. Accordingly, the activityof CARD-3, CARD-4, CARD-5, or CARD-6 protein, expression of CARD-3,CARD-4, CARD-5, or CARD-6 nucleic acid, or mutation content of CARD-3,CARD-4, CARD-5, or CARD-6 genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of CARD-3, CARD-4, CARD-5, or CARD-6 proteinexpression of CARD-3 or CARD-4 nucleic acid, or mutation content ofCARD-3, CARD-4, CARD-5, or CARD-6 genes in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a CARD-3, CARD-4, CARD-5, or CARD-6 modulator, such as a modulatoridentified by one of the exemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of CARD-3, CARD-4, CARD-5, or CARD-6 (e.g., theability to modulate aberrant cell proliferation and/or differentiation)can be applied not only in basic drug screening, but also in clinicaltrials. For example, the effectiveness of an agent determined by ascreening assay as described herein to increase CARD-3, CARD-4, CARD-5,or CARD-6 gene expression, protein levels, or upregulate CARD-3, CARD-4,CARD-5, or CARD-6 activity, can be monitored in clinical trails ofsubjects exhibiting decreased CARD-3, CARD-4, CARD-5, or CARD-6 geneexpression, protein levels, or downregulated CARD-3, CARD-4, CARD-5, orCARD-6 activity. Alternatively, the effectiveness of an agent determinedby a screening assay to decrease CARD-3, CARD-4, CARD-5, or CARD-6 geneexpression, protein levels, or downregulated CARD-3, CARD-4, CARD-5, orCARD-6 activity, can be monitored in clinical trials of subjectsexhibiting increased CARD-3, CARD-4, CARD-5, or CARD-6 gene expression,protein levels, or upregulated CARD-3, CARD-4, CARD-5, or CARD-6activity. In such clinical trials, the expression or activity of CARD-3,CARD-4, CARD-5, or CARD-6 and preferably, other genes that have beenimplicated in, for example, a cellular proliferation disorder can beused as a “read out” or markers of the immune responsiveness of aparticular cell.

For example, and not by way of limitation, genes, including CARD-3,CARD-4, CARD-5, or CARD-6, that are modulated in cells by treatment withan agent (e.g., compound, drug or small molecule) which modulatesCARD-3, CARD-4, CARD-5, or CARD-6 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on cellular proliferation disorders, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of CARD-3, CARD-4, CARD-5, or CARD-6 andother genes implicated in the disorder. The levels of gene expression(i.e., a gene expression pattern) can be quantified by Northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of protein produced, by one of the methods as describedherein, or by measuring the levels of activity of CARD-3, CARD-4,CARD-5, or CARD-6 or other genes. In this way, the gene expressionpattern can serve as a marker, indicative of the physiological responseof the cells to the agent. Accordingly, this response state may bedetermined before, and at various points during, treatment of theindividual with the agent.

In an embodiment, the present invention provides a method for monitoringthe effectiveness of treatment of a subject with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate identified by the screeningassays described herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a CARD-3, CARD-4,CARD-5, or CARD-6 protein, mRNA, or genomic DNA in the preadministrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of theCARD-3, CARD-4, CARD-5, or CARD-6 protein, mRNA, or genomic DNA in thepost-administration samples; (v) comparing the level of expression oractivity of the CARD-3, CARD-4, CARD-5, or CARD-6 protein, mRNA, orgenomic DNA in the pre-administration sample with the CARD-3, CARD-4,CARD-5, or CARD-6 protein, mRNA, or genomic DNA in the postadministration sample or samples; and (vi) altering the administrationof the agent to the subject accordingly. For example, increasedadministration of the agent may be desirable to increase the expressionor activity of CARD-3, CARD-4, CARD-5, or CARD-6 to higher levels thandetected i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of CARD-3, CARD-4, CARD-5, or CARD-6 tolower levels than detected, i.e., to decrease the effectiveness of theagent.

5. Transcriptional Profiling

The CARD-3, CARD-4, CARD-5, and CARD-6 nucleic acid molecules describedherein, including small oligonucleotides, can be used intranscriptionally profiling. For example, these nucleic acids can beused to examine the expression of CARD-3, CARD-4, CARD-5, and CARD-6 innormal tissue or cells and in tissue or cells subject to a diseasestate, e.g., tissue or cells derived from a patient having a disease ofinterest or cultured cells which model or reflect a disease state ofinterest, e.g., cells of a cultured tumor cell line. By measuringexpression of CARD-3, CARD-4, CARD-5, and CARD-6, together orindividually, a profile of expression in normal and disease states canbe developed. This profile can be used diagnostically and to examine theeffectiveness of a therapeutic regime.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant CARD-3, CARD-4, CARD-5, orCARD-6 expression or activity, examples of which are provided herein.

1. Prophylactic Methods

In one aspect the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant CARD-3,CARD-4, CARD-5, or CARD-6 expression or activity, by administering tothe subject an agent which modulates CARD-3, CARD-4, CARD-5, or CARD-6expression or at least one CARD-3, CARD-4, CARD-5, or CARD-6 activity.Subjects at risk for a disease which is caused or contributed to byaberrant CARD-3, CARD-4, CARD-5, or CARD-6 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe CARD-3, CARD-4, CARD-5, or CARD-6 aberrancy, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.Depending on the type of CARD-3, CARD-4, CARD-5, or CARD-6 aberrancy,for example, a CARD-3, CARD-4, CARD-5, or CARD-6 agonist or CARD-3,CARD-4, CARD-5, or CARD-6 antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein. Activities of CARD-3, CARD-4, CARD-5, or CARD-6that could be modulated for prophylactic purposes include, but are notlimited to: 1) CARD-3, CARD-4, CARD-5, or CARD-6 gene or proteinexpression, for example, see Example 11 for a description of the mRNAexpression pattern of human CARD-4; 2)CARD-3, CARD-4, CARD-5, or CARD-6binding to a target protein, for example, see Examples 7, 8, 12, and 17for a description of proteins known to bind to CARD-3, CARD-4, orCARD-5; 3) CARD-4 and CARD-5 regulation of NF-κB as described in Example9 and 18; and 4) CARD-3 and CARD-4 enhancement of caspase 9 activity asdescribed in Example 10.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingCARD-3, CARD-4, CARD-5, or CARD-6 expression or activity for therapeuticpurposes. The modulatory method of the invention involves contacting acell with an agent that modulates one or more of the activities ofCARD-3, CARD-4, CARD-5, or CARD-6 protein activity associated with thecell. An agent that modulates CARD-3, CARD-4, CARD-5, or CARD-6 proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring cognate ligand of a CARD-3, CARD-4,CARD-5, or CARD-6 protein, a peptide, a CARD-3, CARD-4, CARD-5, orCARD-6 peptidomimetic, or other small molecule. In one embodiment, theagent stimulates one or more of the biological activities of CARD-3,CARD-4, CARD-5, or CARD-6 protein. Examples of such stimulatory agentsinclude active CARD-3, CARD-4, CARD-5, or CARD-6 protein and a nucleicacid molecule encoding CARD-3, CARD-4, CARD-5, or CARD-6 that has beenintroduced into the cell. In another embodiment, the agent inhibits oneor more of the biological activities of CARD-3, CARD-4, CARD-5, orCARD-6 protein. Examples of such inhibitory agents include antisenseCARD-3, CARD-4, CARD-5, or CARD-6 nucleic acid molecules andanti-CARD-3, CARD-4, CARD-5, or CARD-6 antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively in vivo (e.g, by administering the agent to asubject). As such the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a CARD-3, CARD-4, CARD-5, or CARD-6protein or nucleic acid molecule or a disorder related to CARD-3,CARD-4, CARD-5 or CARD-6 expression or activity. In one embodiment, themethod involves administering an agent (e.g., an agent identified by ascreening assay described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) CARD-3, CARD-4, CARD-5,or CARD-6 expression or activity. In another embodiment, the methodinvolves administering a CARD-3, CARD-4, CARD-5, or CARD-6 protein ornucleic acid molecule as therapy to compensate for reduced or aberrantCARD-3, CARD-4, CARD-5, or CARD-6 expression or activity. Activities ofCARD-3, CARD-4, CARD-5, or CARD-6 that could be modulated fortherapeutic purposes include, but are not limited to, 1) CARD-3, CARD-4,CARD-5, or CARD-6 gene or protein expression, for example, see Example11 for a description of the mRNA expression pattern of human CARD-4;2)CARD-3, CARD-4, CARD-5, or CARD-6 binding to a target protein, forexample, see Examples 7, 8, 12, and for a description of proteins knownto bind to CARD-3, CARD-4, or CARD-5; 3) CARD-4 or CARD-5 regulation ofNF-κB as described in Examples 9 and 18; and 4) CARD-4 enhancement ofcaspase 9 activity as described in Example 10.

Stimulation of CARD-3, CARD-4, CARD-5, or CARD-6 activity is desirablein situations in which CARD-3, CARD-4, CARD-5, or CARD-6 is abnormallydownregulated and/or in which increased CARD-3, CARD-4, CARD-5, orCARD-6 activity is likely to have a beneficial effect. Conversely,inhibition of CARD-3, CARD-4, CARD-5, or CARD-6 activity is desirable insituations in which CARD-3, CARD-4, CARD-5, or CARD-6 is abnormallyupregulated, e.g., in myocardial infarction, and/or in which decreasedCARD-3, CARD-4, CARD-5, or CARD-6 activity is likely to have abeneficial effect. Since CARD-4 and CARD-5 may be involved in theprocessing of cytokines, inhibiting the activity or expression CARD-4 orCARD-5 may be beneficial in patients that have aberrant inflammation.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLES Example 1 Isolation and Characterization of Full-Length HumanCARD-3 and CARD-4L/S cDNAs

A profile of known CARD domains was used to search databases of cDNAsequences and partial cDNA sequences using TBLASTN (WashingtonUniversity; version 2.0, BLOSUM62 search matix). This search led to theidentification of CARD-3. Using CARD-3 to search databases of cDNAsequences and partial cDNA sequences, another potential CARD cDNA wasfound. This cDNA sequence was used screen a human umbilical veinendothelial library (HUVE) and a clone containing the partial CARD-4Swas identified. The human umbilical vein endothelial library was thenrescreened using a probe designed against the partial CARD-4S sequenceand a clone containing the CARD-4L sequence was identified.

Example 2 Characterization of CARD-3 AND CARD-4L/S Proteins

In this example, the predicted amino acid sequences of human CARD-3 andCARD-4L/S proteins were compared to amino acid sequences of knownproteins and various motifs were identified. For example, the CARDdomains of CARD-3 and CARD-4 were aligned (FIG. 7) with the CARD domainsof ARC-CARD (SEQ ID NO:31), cIAP1-CARD (SEQ ID NO:32) and cIAP2-CARD(SEQ ID NO:33). In addition, the molecular weight of the human CARD-3and CARD-4L/S proteins were predicted.

The human CARD-3 cDNA was isolated as described above (FIG. 1; SEQ IDNO:1) and encodes a 540 amino acid protein (FIG. 2: SEQ ID NO:2). CARD-3also >includes one predicted kinase domain (amino acid 1 to amino acid300 of SEQ ID NO:2; SEQ ID NO:4), which is followed by a predictedlinker domain (amino acid 301 to amino acid 431 of SEQ ID NO:2; SEQ IDNO:5) and a predicted CARD domain (amino acid 432 to amino acid 540 ofSEQ ID NO:2; SEQ ID NO:6).

The human CARD-4L cDNA was isolated as described above (FIG. 3; SEQ IDNO:7) and has a 2859 nucleotide open reading frame (nucleotides 245-3103of SEQ ID NO:7; SEQ ID NO:9) which encodes a 953 amino acid protein(FIG. 4; SEQ ID NO:8). CARD-4L protein has a predicted CARD domain(amino acids 15-114; SEQ ID NO: 10). CARD-4L is also predicted to have anucleotide binding domain which extends from about amino acid 198 toabout amino acid 397 of SEQ ID NO:8; SEQ ID NO:11, a predicted WalkerBox “A”, which extends from about amino acid 202 to about amino acid 209of SEQ ID NO:8; SEQ ID NO:12, a predicted Walker Box “B”, which extendsfrom about amino acid 280 to about amino acid 284, of SEQ ID NO:8; SEQID NO:13, a predicted kinase 1a (P-loop) domain, which extends fromabout amino acid 197 to about amino acid 212 of SEQ ID NO:8; SEQ IDNO:46, a predicted kinase 2 domain, which extends from about amino acid273 to about amino acid 288 of SEQ ID NO:8; SEQ ID NO:47, a predictedkinase 3a subdomain, which extends from about amino acid 327 to aboutamino acid 338 of SEQ ID NO:8; SEQ ID NO:14, ten predicted Leucine-richrepeats which extend from about amino acid 674 to about amino acid 950of SEQ ID NO:8. The first Leucine-rich repeat is predicted to extendfrom about amino acid 674 to about amino acid 701 of SEQ ID NO:8; SEQ IDNO:15. The second Leucine-rich repeat is predicted to extend from aboutamino acid 702 to about amino acid 727 of SEQ ID NO:8; SEQ ID NO:16. Thethird Leucine-rich repeat is predicted to extend from about amino acid728 to about amino acid 754 of SEQ ID NO:8; SEQ ID NO: 17. The fourthLeucine-rich repeat is predicted to extend from about amino acid 755 toabout amino acid 782 of SEQ ID NO:8; SEQ ID NO:18. The fifthLeucine-rich repeat is predicted to extend from about amino acid 783 toabout amino acid 810 of SEQ ID NO:8; SEQ ID NO:19. The sixthLeucine-rich repeat is predicted to extend from about amino acid 811 toabout amino acid 838 of SEQ ID NO:8; SEQ ID NO:20. The seventhLeucine-rich repeat is predicted to extend from about amino acid 839 toabout amino acid 866 of SEQ ID NO:8; SEQ ID NO:21. The eighthLeucine-rich repeat is predicted to extend from about amino acid 867 toabout amino acid 894 of SEQ ID NO:8; SEQ ID NO:22. The ninthLeucine-rich repeat is predicted to extend from about amino acid 895 toabout amino acid 922 of SEQ ID NO:8; SEQ ID NO:23 and the tenthleucine-rich repeat is predicted to extend from about amino acid 923 toabout amino acid 950 of SEQ ID NO:8; SEQ ID NO:24.

The human partial CARD-4S cDNA isolated as described above (FIG. 5; SEQID NO:25) encodes a 490 amino acid protein (FIG. 6; SEQ ID NO:26).CARD-4S includes one predicted partial CARD domain (amino acids 1-74 ofSEQ ID NO:26). CARD-4S is also predicted to have a P-Loop which extendsfrom about amino acid 163 to about amino acid 170 of SEQ ID NO:26; SEQID NO:29, and a predicted Walker Box “B” which extends form about aminoacid 241 to about amino acid 245 of SEQ ID NO:26; SEQ ID NO:30.

A plot showing the predicted structural features of CARD-4L is presentedin FIG. 8. This figure shows the predicted alpha regions(Gamier-Robinson and Chou-Fasman), the predicted beta regions(Gamier-Robinson and Chou-Fasman), the predicted turn regions(Garnier-Robinson and Chou-Fasman) and the predicted coil regions(Garnier-Robinson and Chou-Fasman). Also included in the figure is ahydrophilicity plot (Kyte-Doolittle), the predicted alpha andbeta-amphatic regions (Eisenberg), the predicted flexible regions(Karplus-Schulz), the predicted antigenic index (Jameson-Wolf) and thepredicted surface probability plot (Emini).

A plot showing the predicted sturctural features of CARD-4S is alsopresented in FIG. 9. This figure shows the predicted alpha regions(Gamier-Robinson and Chou-Fasman), the predicted beta regions(Gamier-Robinson and Chou-Fasman), the predicted turn regions(Gamier-Robinson and Chou-Fasman) and the predicted coil regions(Gamier-Robinson and Chou-Fasman). Also included in the figure is ahydrophilicity plot (Kyte-Doolittle), the predicted alpha andbeta-amphatic regions (Eisenberg), the predicted flexible regions(Karplus-Schulz), the predicted antigenic index (Jameson-Wolf) and thepredicted surface probability plot (Emini).

The predicted MW of CARD-3 is approximately 61 kDa. The predicted MW ofCARD-4L is approximately 108 kDa.

Example 3 Preparation of CARD-3 and CARD-4 Proteins

Recombinant CARD-3 and CARD-4 can be produced in a variety of expressionsystems. For example, the CARD-3 and CARD-4 peptides can be expressed asa recombinant glutathione-S-transferase (GST) fusion protein in E. coliand the fusion protein can be isolated and characterized. Specifically,as described above, CARD-3 or CARD-4 can be fused to GST and the fusionprotein can be expressed in E. coli strain PEB199. Expression of theGST-CARD-3 or GST-CARD-4 fusion protein in PEB199 can be induced withIPTG. The recombinant fusion protein can be purified from crudebacterial lysates of the induced PEB 199 strain by affinitychromatography on glutathione beads.

Example 4 Identification of Splice Variants of CARD-4

The 5′ untranslated sequence from CARD-4L was used to search databasesof cDNA sequences and partial cDNA sequences using BLASTN (WashingtonUniversity; version 2.0, BLOSUM62 search matrix) for additional CARD-4cDNA clones. This search led to the identification of two cDNA clones,clone Z from a human lymph node library and the Y clone from a humanbrain cDNA library. Both clones were sequenced and found to representprobable splice variants of CARD-4 that encode truncated CARD-4proteins, Y encoding a 249 amino acid protein and Z encoding a 164 aminoacid protein. FIG. 10 shows the nucleotide (SEQ ID NO:38) and FIG. 11the predicted amino acid (SEQ ID NO:39) sequences of human CARD-4Y; FIG.12 shows the nucleotide (SEQ ID NO:40) and FIG. 13 the amino acid (SEQID NO:41) sequences of human CARD-4Z; and FIG. 14 shows an alignment ofthe CARD-4L. CARD-4Y, and CARD-4Z amino acid sequences generated by theClustal program using a PAM250 residue weight table.

Example 5 Identification of Murine CARD-4

The CARD-4 polypeptide sequence was used to search databases of cDNAsequences and partial cDNA sequences using the TBLASTN program (version1.4, BLOSUM62 search matrix, and a word length of 3) for murine CARD-4cDNA clones. This search led to the identification of a partial murineCARD-4 clone designated murine CARD-4L. The rapid identification of cDNAends procedure (RACE) was applied to the 5′ end of the murine CARD-4Lclone to elucidate the 5′ end of the murine CARD-4L cDNA. FIG. 15 showsthe murine CARD-4L nucleotide sequence(SEQ ID NO:42), FIG. 16 shows themurine CARD-4L amino acid sequence (SEQ ID NO:43), and FIG. 17 shows analignment of the murine CARD-4L and human CARD-4L amino acid sequencesgenerated by the Clustal program using a PAM250 residue weight table.

Example 6 Identification of the Chromosomal Location of Human CARD-4

To determine the chromosomal location of the human CARD-4 gene, thepolymerase chain reaction carried out with human CARD-4-specific primerscard4t, with the 5′ to 3′ sequence agaaggtctggtcggcaaa (SEQ ID NO:44),and card4k, with the 5′ to 3′ sequence aagccctgagtggaagca (SEQ IDNO:45), was used to screen DNAs from a commercially available somaticcell hybrid panel. This analysis showed that human CARD-4 maps tochromosome 7 close to the SHGC-31928 genetic marker.

Example 7 Identification of CARD-3 in a Yeast Two-Hybrid Screen forProteins that Physically Interact with the CARD Domain of Human CARD-4

DNA encoding amino acids 1-145 of human CARD-4 comprising the CARDdomain was cloned into a yeast two-hybrid screening vector to create aCARD-4,1-145-GAL4 DNA-binding domain fusion for two-hybrid screening.The CARD-4,1-145-GAL4 DNA-binding domain fusion was used to screen humanmammary gland and human prostate two-hybrid libraries for gene productsthat could physically associate with CARD-4,1-145. Twelve libraryplasmids expressing CARD4,1-145 interacting proteins were found tocontain the CARD-domain containing protein CARD-3 thus establishing adirect or indirect physical interaction between CARD-4 and CARD-3.

In addition, DNA encoding amino acids 435-540 of CARD-3 comprising theCARD domain of CARD-3 (SEQ ID NO:6) was cloned into a yeast two-hybridGAL4 transcriptional activation domain fusion vector to create aCARD-3,435-540-GAL4 transcriptional activation domain fusion. To testwhether the CARD domain of CARD-3 binds CARD-4,1-145, theCARD-3,435-540-GAL4 transcriptional activation domain fusion expressionvector and the CARD-4,1-145-GAL4 DNA-binding domain fusion vector werecotransformed into a two-hybrid screening Saccharomyces cerevisiae(yeast) strain. The resulting cotransformed yeast strain expressed thetwo reporter genes that indicate a physical interaction between the twohybrid proteins in the experiment, in this case, the CARD-3,435-540-GAL4transcriptional activation domain fusion protein and theCARD-4,1-145-GAL4 DNA-binding domain fusion protein. This experimentestablished a physical interaction between the CARD domain of CARD-3 andthe CARD domain of CARD-4.

Example 8 Identification of hNUDC in a Yeast Two-Hybrid Screen forProteins that Physically Interact with the LRR Domain of Human CARD-4

DNA encoding amino acids 406-953 of human CARD-4L comprising the LRRdomain was cloned into a yeast two-hybrid screening vector to create aCARD-4,406-953-GAL4 DNA-binding domain fusion for two-hybrid screening.The CARD-4,406-953-GAL4 DNA-binding domain fusion was used to screen ahuman mammary gland two-hybrid library for gene products that couldphysically associate with CARD-4,406-953. One library plasmid expressinga CARD-4,406-953 interacting protein was found to contain the hNUDCprotein, the human ortholog of the rat NUDC protein that has beenimplicated in nuclear movement (Morris et al., Curr. Biol. 8:603 [1998],Morris et al., Exp. Cell Res. 238:23 [1998]), thus establishing aphysical interaction between CARD-4 and hNUDC.

Example 9 Discovery of Regulation by CARD-4 of NF-κB

The first group of experiments described in this Example were carriedout to determine if CARD-4 can activate the NF-κB pathway. CARD-4regulation of the NF-κB pathway is of interest because the NF-κB pathwayis involved in many diseases described in (New England Journal ofMedicine 336:1066 [1997]) and (American Journal of Cardiology 76:18C[1995]) and other references known to those skilled in the art.Participation of CARD-4 in the NF-κB pathway would make CARD-4 anattractive target for drugs that modulate the NF-κB pathway fortreatment of NF-κB pathway-dependent diseases, conditions, andbiological processes.

The first group of experiments showed specific CARD-4-mediated NF-κBpathway induction.

The second group of experiments described in this Example were carriedout to determine if CARD-3, the NIK serine/threonine protein kinase (Suet al., EMBO J. 16:1279 [19971), or the signal transduction proteinTRAF6 (Cao et al., Nature 383:443 [1996]), proteins known to participatein the induction of NF-κB (McCarthy et al., J. Biol. Chem. 273:16968[1998]), are involved in transducing the CARD-4-dependent NF-κB pathwayinduction signal. It was found that CARD-3, NIK, and TRAF6 are allinvolved in transducing the CARD-4-mediated NF-κB pathway inductionsignal.

In nine transfection experiments, 293T cells coexpressing an NF-κBreporter plasmid and either pCI, pCI-CARD-4L (expressing CARD-4L),pCI-CARD-4S (expressing CARD-4S), pCI-APAFL (expressing Apaf-1),pCI-APAFS (expressing an Apaf-1 variant lacking WD repeats),pCI-CARD-4LnoCARD (expressing CARD-4L without a CARD domain),pCI-CARD4LnoLRR (expressing CARD-4L without a LRR), pCI-CARD4LCARDonly(expressing CARD-4L CARD domain only), or pCI-CARD4NBSonly (expressingCARD-4L nucleotide binding sequence only) were created. 293T cells cellswere plated in 6-well plates (35 mm wells) and transfected 2 days later(90% confluency) with 1 μg of NF-κB luciferase reporter plasmid(pNF-κB-Luc, Stratagene). 200 ng of pCMV β-gal, 600 ng of pCI vector and200 ng of indicated expression plasmids using SuperFect transfectionreagent (Qiagen). For dominant-negative experiments, 2 ng of CARD4expressing plasmid and 800 ng of dominant-negative plasmid were used.Cells were harvested 48 h after transfection and luciferase activity in1000-fold diluted cell extracts was determined using the LuciferaseAssay System (Promega). In addition, β-galactosidase activities weredetermined and used to normalize transfection efficiency.

Relative luciferase activity was determined at the end of the experimentto assess NF-κB pathway activation by the gene expressed by thepCI-based plasmid in each transfected cell line. The cell linescontaining pCI, pCI-APAFS, pCI-APAFL, pCI-CARD-4LnoCARD, andpCI-CARD4NBSonly had similar baseline levels of luciferase expressionbut the cell lines containing pCI-CARD-4L, pCI-CARD4LnoLRR, andpCI-CARD4LCARDonly had luciferase expression about nine fold elevatedrelative to baseline and the cell line containing pCI-CARD4S hadluciferase expression sixteen fold elevated relative to baseline. Thisresult demonstrates induction by CARD-4S and CARD-4L of the NF-κBpathway. This CARD-4 mediated NF-κB pathway induction is dependent onthe CARD-4 CARD domain because the pCI-CARD-4noCARD construct expressingCARD-4 lacking its CARD domain did not induce the luciferase reportergene and pCI-CARD4LCARDonly expressing the CARD-4 CARD domain did inducethe luciferase reporter gene. Also, the CARD-4 LRR domains are notrequired for NF-κB pathway activation because pCI-CARD4LnoLRR expressinga CARD-4 mutant protein lacking LRR domains is able to induce theluciferase reporter gene. In addition, the CARD-4 NBS domain is notsufficient for NF-κB pathway activation because pCI-CARD4NBSonlyexpressing CARD-4 NBS domain is not able to induce the luciferasereporter gene. In addition, the induction of the NF-κB pathway by CARD-4is specific, as neither Apaf-expressing construct in this experimentinduced luciferase activation.

In five transfection experiments, 293T cells coexpressing an NF-κBreporter plasmid (NF-κB-luciferase, Stratagene) and pCI-CARD-4L andeither, no vector, pCI-TRAF6-DN (expressing a dominant negative versionof TRAF-6), pCI-NIK-DN (expressing a dominant negative version of NIKkinase), pCI-CARD3CARDonly (expressing the CARD domain of CARD-3, whichacts as a dominant negative version of CARD-3), or pCI-Bcl-XL(expressing the anti-apoptotic protein Bcl-XL) were created. TRAF6-DN,NIK-DN, and CARD3-CARDonly are dominant negative alleles of the TRAF6,NIK, and CARD3 genes, respectively. After 48 hours, cells were lysed andthe relative luciferase activity was determined (Promega Kit) to assessNF-κB pathway activation by the genes expressed by the one or twopCI-based plasmids in each transfected cell line. The cell linescontaining pCI-CARD-4L only or pCI-CARD-4L and pCI-Bcl-XL had relativeluciferase reporter gene expression of about 18 units. The cell linescontaining pCI-CARD-4L and pCI-TRAF6-DN, pCI-CARD-4L and pCI-NIK-DN, orpCI-CARD-4L and pCI-CARD3CARDonly had relative luciferase reporter geneexpression of about 4 units. Inhibition of CARD-4L-mediated NF-κBpathway induction by TRAF6-DN, NIK-DN, and CARD-3CARDonly is specific asBcl-XL did not inhibit CARD-4L-mediated NF-κB pathway induction.

These results demonstrate that dominant negative alleles of TRAF6, NIKand CARD-3 expressed, respectively, from pCI-TRAF6-DN, pCI-NIK-DN, andpCI-CARD3CARDonly block induction of the NF-κB reporter gene by CARD-4Lexpression (pCI-CARD-4L) and suggest that TRAF6, NIK, and CARD-3 actdownstream of CARD-4L to transduce the CARD-4L NF-κB pathway inductionstimulus.

In an additional experiment, coexpression of CARD-4 and the CARD domainof CARD-3 revealed that the CARD domain of CARD-3 functions as adominant negative mutant suggesting that CARD-3 is a downstream mediatorof CARD-4 function.

Example 10 Discovery of CARD-4 Enhancement of Caspase-9 Activity

In ten transfection experiments, 293T cells coexpressing a betagalactosidase-expressing plasmid (pCMV β-gal from Stratagene) as amarker for viable cells and either pCI, pCI-CARD-3, pCI-APAF,pCI-CARD-4L, pCI-CARD-4S, pCI-CARD4LnoLRR, pCI-CARD4NBSonly,pCI-CARD4LCARDonly, pCI-CARD-4LnoCARD or pCI-casp9 (expressingcaspase-9) were created. Transfections included 400 ng of pCMV P-gal,800 ng of expression plasmid, and Superfect transfection reagent fromQiagen and were carried out according to the manufacturer's directions.After 40-48 hours, cells were fixed and stained for beta-galactosidaseexpression and cell viability was determined by counting the number ofbeta galactosidase positive cells. Expression of pCI, pCI-CARD-3,pCI-APAF, pCI-CARD-4L, pCI-CARD-4S, pCI-CARD4LnoLRR, pCI-CARD4NBSonly,pCI-CARD4LCARDonly, and pCI-CARD-4LnoCARD did not result in loss of cellviability. As expected, expression of pCI-casp9 in 293T cells resultedin a loss of viability of about 75% of the cells in the experiment.

It was next tested whether pCI, pCI-CARD-3, pCI-APAF, pCI-CARD-4L,pCI-CARD-4S, pCI-CARD4LnoLRR, pCI-CARD4NBSonly, pCI-CARD4LCARDonly, orpCI-CARD-4LnoCARD would regulate caspase 9-mediated apoptosis. In ninetransfection experiments. 293T cells coexpressing a betagalactosidase-expressing plasmid as a marker for viable cells,pCI-casp9, and either pCI, pCI-CARD-3, pCI-APAF, pCI-CARD-4L,pCI-CARD-4S, pCI-CARD4LnoLRR, pCI-CARD4NBSonly, pCI-CARD4LCARDonly, andpCI-CARD-4LnoCARD were created. After 40-48 hours, cells were fixed andstained for beta-galactosidase expression and cell viability wasdetermined by counting the number of beta galactosidase positive cells.Expression of pCI, pCI-CARD-4LnoCARD, and pCI-CARD4NBSonly in thecaspase 9-expressing 293T cells had no effect on the caspase 9-inducedapoptosis. However, pCI-CARD-3, pCI-CARD-4L, pCI-CARD-4S,pCI-CARD4LnoLRR, pCI-CARD4LCARDonly and, as expected, pCI-APAF enhancedthe level of caspase 9-induced apoptosis to 20 or less betagalactosidase positive cells per experiment from about 100 betaglactosidase positive cells per experiment.

This experiment demonstrated that CARD-4 can enhance caspase 9-mediatedapoptosis because coexpression of CARD-4L or CARD-4S with caspase-9dramatically increases caspase-9 mediated apoptosis. Furthermore, theCARD-4 CARD domain (SEQ ID NO:10) is necessary and sufficient forCARD-4-mediated enhancement of caspase-9-potentiated apoptosis becauseCARD-4L lacking its CARD domain (pCI-CARD-4LnoCARD) does not enhancecaspase-9-mediated apoptosis while the CARD-4 CARD domain expressedalone (pCI-CARD4LCARDonly) does induce caspase-9 mediated apoptosis. Inaddition, the LRR present in CARD-4 is not required for CARD-4enhancement of caspase-9-mediated apoptosis because expression of aCARD-4 protein lacking the LRR (pCI-CARD4LnoLRR) still enhancescaspase-9-mediated apoptosis. The CARD-4 NBS is not sufficient forCARD-4 enhancement of caspase-9-mediated apoptosis because expression ofthe CARD-4 NBS only (pCI-CARD4NBSonly) does not enhance caspase-9mediated apoptosis. This experiment also demonstrates that CARD-3 canenhance caspase-9-mediated apoptosis.

As detailed below in Example 12, CARD-4 does not appear to interactdirectly with caspase-9, suggesting that potentiation of caspase-9activity by CARD-4 is mediated by activation of downstream pathways.

Example 11 Identification and Tissue Distribution of mRNA SpeciesExpressed by the Human CARD-4 Gene

Northern analysis of mRNAs extracted from adult human tissues revealed a4.6 kilobase mRNA band that was expressed in most tissues examined.Highest expression was observed heart, spleen, placenta and lung. CARD-4was also observed to be expressed in fetal brain, lung, liver andkidney. Cancer cell lines expressing the 4.6 kilobase CARD-4 mRNAinclude HeLa, K562, Molt4, SW480, A549 and melanoma. A larger 6.5 to 7.0kilobase CARD-4 mRNA was expressed in heart, spleen, lung, fetal lung,fetal liver, and in the Molt4 and SW480 cell lines.

Example 12 Physical Association of CARD-4 with CARD-3

CARD-4-specific PCR primers with the 3′ primer encoding the HA epitopetag were used to amplify the CARD-4L gene epitope tagged with HA andthis PCR product was cloned into the mammalian expression vector pCI.CARD-3-specific PCR primers with the 5′ primer encoding the MYC epitopetag were used to amplify the CARD-3 gene epitope tagged with MYC andthis PCR product was cloned into the mammalian expression vector pCI.CARD-3-specific PCR primers with the 5′ primer encoding the MYC epitopetag were used to amplify the CARD-3 gene lacking the CARD domain (SEQ IDNO:6) epitope tagged with MYC and this PCR product was cloned into themammalian expression vector pCI. Caspase 9-specific PCR primers with the3′ primer encoding the MYC epitope tag were used to amplify the caspase9 gene epitope tagged with MYC and this PCR product was cloned into themammalian expression vector pCI. In three transfection experiments, 293Tcells coexpressing pCI-CARD-4LcHA and either pCI-CARD3nMYC,pCI-CARD3noCARDnMYC, or pCI-casp9cMYC were created. Cells from eachtransfected line were lysed and an immunoprecipitation procedure wascarried out on each lysate with an anti-MYC epitope tag antibody toprecipitate the CARD-4LcHA expressed by each cell line and anyphysically associated proteins. Immunoprecipitated proteins wereseparated by electrophoresis on denaturing polyacrylamide gels,transferred to nylon filters, and probed with an anti-HA epitope tagantibody in a Western blotting experiment to determine whether theMYC-tagged protein that was coexpressed with the CARD-4LcHA protein hadcoimmunoprecipitated with the CARD-4LcHA protein. In this experiment.CARD-3 was found to coimmunoprecipitate with CARD-4 while CARD-3 lackingits CARD domain and caspase-9 did not coimmunoprecipitate with CARD-4.This experiment demonstrates that CARD-4 and CARD-3 physically associateand that CARD-3 requires its CARD domain to associate with CARD-4. Inaddition, CARD-4 appears to not associate with caspase-9.

Example 13 CARD-4 Genomic Sequence

FIG. 18 is depicts the 32042 nucleotide genomic sequence of CARD-4 (SEQID NO:63). This sequence is based the CARD-4 cDNA sequence describedabove and a BAC sequence (DBEST Accession No. AC006027). The CARD-4 cDNAsequence described above was used to correct three errors in the BACsequence, including one error resulting in a frameshift. The CARD-4genomic sequence of FIG. 18 includes the following introns and exons:exon 1: nucleotides 364-685, encoding amino acids 1-67 (start codon atnucleotides 485-487); intron 1: nucleotides 686-2094; exon 2:nucleotides 2095-2269, encoding amino acids 67-126; intron 2:nucleotides 2270-4365; exon 3: nucleotides 366-6190, encoding aminoacids 126-734; intron 3: nucleotides 6191-9024; exon 4: nucleotides9025-9108, encoding amino acids 734-762; intron 4: nucleotides9109-10355; exon 5: nucleotides 10356-10439, encoding amino acids762-790; intron 5: nucleotides 10440-11181; exon 6: nucleotides1182-11265, encoding amino acids 790-818; intron 6: nucleotides11266-19749; exon 7: nucleotides 19750-19833, encoding amino acids818-846; intron 7: nucleotides 19834-21324; exon 8: nucleotides21325-21408, encoding amino acids 846-874; intron 8: nucleotides21409-24226; exon 9: nucleotides 24227-24310, amino acids 874-903;intron 9: nucleotides 24311-27948; exon 10: nucleotides 27949-28032,amino acids 903-930; intron 10: nucleotides 28033-31695; exon I 1:nucleotides 31696-32024, encoding amino acids 930-953 (stop codon atnucleotides 31766-31768).

The introns in the CARD-4 genomic sequence contain consensus splicedonor and acceptor sites (Molecular Cell Biology, Darnell et al., eds.,1996). The CARD-4 genomic sequence is usful for genetic identificationand mapping and identifying mutations, e.g., mutations is splice donoror splice acceptor sites.

Example 14 Isolation and Characterization of Full-Length Murine CARD-5and Human CARD-5

The amino acid sequence of the CARD domain of RAIDD (amino acids 1 to94) was used to search a proprietary murine cDNA sequence database usingthe BLASTX program with the BLOSUM62 matrix and a protein word length ofthree. This search led to the identification of a murine clone,jtmaa010ht2, present in a coronary artery smooth muscle cell library.This clone encodes a protein designated CARD-5. The 761 nucleotidemurine CARD-5 cDNA of SEQ ID NO:60 has a 579 nucleotide open readingframe (SEQ ID NO:62) encoding a 193 amino acid protein (SEQ ID NO:61).The cDNA and protein sequences of murine CARD-5 are shown in FIG. 19.

Murine CARD-5 is predicted to be an intracellular protein having amolecular weight of 21.4 kDa prior to post-translational modification.

FIG. 20 depicts a hydropathy plot of murine CARD-5. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and potential N-glycosylation sites (Ngly)are indicated by short vertical lines just below the hydropathy trace.

The murine CARD-5 nucleotide sequence was used to search a proprietarydatabase of human cDNA sequences. This search led to the identificationof a human CARD-5 cDNA clone, jthza027g11t1, present in a testeslibrary.

The 740 nucleotide murine CARD-5 cDNA of SEQ ID NO:48 has a 585nucleotide open reading frame (SEQ ID NO:50) encoding a 195 amino acidprotein (SEQ ID NO:49). The cDNA and protein sequences of human CARD-5are shown in FIG. 21.

Human CARD-5 is predicted to be an intracellular protein having amolecular weight of 21.6 kDa prior to post-translational modification.

FIG. 22 depicts a hydropathy plot of human CARD-5. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and potential N-glycosylation sites (Ngly)are indicated by short vertical lines just below the hydropathy trace.

FIG. 23 depicts an alignment of the cDNA sequences of murine (SEQ IDNO:60) and human (SEQ ID NO:48) CARD-5. In this alignment the sequencesare 68.2% identical. FIG. 24 depicts an alignment of the amino acidsequences of murine (SEQ ID NO:61) and human (SEQ ID NO:49) CARD-5. Inthis alignment the sequences are 71.8% identical.

Both murine and human CARD-5 include a CARD domain. The CARD domain ofmurine CARD-5 extends from amino acid 110 to 179 of SEQ ID NO:61 (SEQ IDNO:57). The CARD domain of human CARD-5 extends from amino acid 111 to181 of SEQ ID NO:49 (SEQ ID NO:58). FIG. 27 depicts an alignment of theCARD domains of murine CARD-5 (SEQ ID NO:57), human CARD-5 (SEQ IDNO:58), and RAIDD (SEQ ID NO:70).

Example 15 Isolation and Characterization of Full-Length Rat CARD-6 andHuman CARD-6

A generalized CARD domain model was used to search a proprietary ratcDNA sequence database. This search led to the identification of a ratcDNA clone present in a sciatic nerve cDNA library. This clone encodes aprotein desigated CARD-6. The 5252 nucleotide rat CARD-6 cDNA of SEQ IDNO:51 has a 2715 nucleotide open reading frame (SEQ ID NO:53) encoding a905 amino acid protein (SEQ ID NO:52). The cDNA and protein sequences ofrat CARD-6 are shown in FIG. 25. Rat CARD-6 is predicted to be anintracellular protein having a molecular weight of 100.2 kDa prior topost-translational modification.

FIG. 26 depicts a hydropathy plot of rat CARD-6. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and potential N-glycosylation sites (Ngly) are indicatedby short vertical lines just below the hydropathy trace.

Rat CARD-6 contains a CARD domain which extends from amino acid 1 toamino acid 108 of SEQ ID NO:52 (SEQ ID NO:59). Rat CARD-6 also has aproline-rich c-terminus which extends from amino acid 698 to amino acid905 of SEQ ID NO:52 (SEQ ID NO:65). This proline-rich domain includesfive putative SH3 binding sites. These binding sites have the sequencePXXP and are located at amino acids 710 to 713 (PAHP), 806 to 809(PLRP), 819 to 822 (PIPP), 857 to 860 (PPHP), and 881 to 884 (PSQP) ofSEQ ID NO:52.

The rat CARD-6 cDNA sequence described above was used to search aproprietary sequence database. This search led to the identification ofa clone from a human muscle cell library encoding a carboxy-terminalportion of human CARD-6. A probe designed based on the sequence of thisclone was used to screen a human adrenal gland library. This screeningled to the identification of a clone encoding an amino-terminal portionof human CARD-6. The clone encoding an amino terminal portion of humanCARD-6 contains a region encoding a CARD domain. This CARDdomain-encoding sequence was used to screen a proprietary database. Thisscreening led to the identification of a clone, jthAb086d02, present inan adrenal gland library, which encodes full length human CARD-6.

The 4244 nucleotide human CARD-6 cDNA of SEQ ID NO:54 has a 3111nucleotide open reading frame (SEQ ID NO:56) encoding a 1037 amino acidprotein (SEQ ID NO:55). The cDNA and protein sequences of human CARD-6are shown in FIG. 28.

N-glycosylation sites are present at amino acids 49-52, 415-418, and812-815 of SEQ ID NO:55. Human CARD-6 contains cAMP and cGMP-dependentprotein kinase phosphorylation sites at amino acids 151-154 and 429-432of SEQ ID NO:55. Protein kinase C phosphorylation sites are present atamino acids 34-36, 57-59, 135-137, 154-156, 161-163, 298-300, 339-341,346-348, 443-445, 664-666, 693-695, 746-748, 882-884, 905-907 and951-953 of SEQ ID NO:55. Casein kinase II phosphorylation sites arepresent at amino acids 6-9, 28-31, 40-43, 112-115. 135-138, 154-157,278-281, 321-324, 339-342, 354-357, 642-645, 670-673, and 707-710 of SEQID NO:55. Tyrosine kinase phosphorylation sites are present at aminoacids 37-34 and 163-169 of SEQ ID NO:55. An ATP/GTP-binding site motif A(P-loop) site is present at amino acids 775-782 of SEQ ID NO:55.

FIG. 29 depicts a hydropathy plot of human CARD-6. Relativelyhydrophobic regions are above the horizontal line, and relativelyhydrophilic regions are below the horizontal line. Cysteine residues areindicated by short vertical lines just below the hydropathy trace.

Human CARD-6 is predicted to have a molecular weight of 116.5 kD beforepost-translational modification.

Human CARD-6 includes a CARD domain at amino acids 5-92 of SEQ ID NO:55(SEQ ID NO:64). FIG. 30 depicts an alignment of the CARD domain domainof human CARD-6 and a consensus CARD domain derived from a hidden Markovmodel (SEQ ID NO:67).

Northern blot analysis of rat CARD-6 expression revealed that CARD-6 isexpressed at a high level in the heart (6.5 kb transcript and a 7 kbtranscript). This analysis also revealed that human CARD-6 is expressedin the brain, spleen, lung, liver, muscle and kidney.

Example 16 CARD-6 Increases Intracellular Signaling

The studies described in this Example demonstrate that CARD-6 expressioncan increase intracellular signalling.

In a first study, a vector which expresses rat CARD-6 under the controlof a CMV promoter was transiently transfected into 293 cells along withpNFκβ-Luc (Stratagene Inc., LaJolla, Calif.). The pNFκβ-Luc vector is areporter plasmid in which a luciferase gene is under the control of apromoter which includes a TATA box and five NFκβ binding elements.Cotransfection of the rat CARD-6 expression vector increased luciferaseexpression by pNκβ-Luc 18-fold over that observed in the absence of therat CARD-6 expression vector. This result indicates that CARD-6stimulates a signalling pathway involving NF-κβ.

In a second study, a vector expressing CARD-6 under the control of theCMV promoter was transiently transfected into 293 cells along withpAP-1-Luc (Strategene, Inc.). The pAP-1-Luc vector is a reported plasmidin which a luciferase gene is under the control of a promoter whichincludes a TATA box and seven AP-1 binding sites. Co-transfection of therat CARD-6 expression vector increased luciferase expression bypAP-1-Luc 4-fold over that observed in the absence of the rat CARD-6expression vector. This result indicates that CARD-6 stimulates asignaling pathway involving AP-1.

Additional studies suggest that CARD-6 can stimulate phosphorylation ofCHOP (GADD153), possibly by activating the stress activated kinase,JNK/p38.

Example 17 Identification of Interactions Between CARD-5 and Proteinswith CARD Domains

A mammalian two-hybrid screening assay revealed that CARD-5 interactswith the CARD domain of several CARD domain-containing proteins.

The Stratagene® Mammalian Two-Hybrid Assay Kit (Stratagene, Inc; LaJolla, Calif.) was used to prepare a vector expressing a protein(Gal4-BD/CARD-5) consisting of the DNA binding domain of yeast Gal4(amino acids 1-147) fused to the CARD domain of human CARD-5 (aminoacids 92 to 195 of SEQ ID NO:49). For a description of the Stratagene®Mammalian Two-Hybrid Assay Kit, see, e.g., Hosfield and Chang (1999)Strategies Newsletter 2(2):62-65. In addition, a library of DNAsequences encoding 26 CARD domains was used to create a library ofexpression vectors encoding the murine NF-κB transcriptional activationdomain (amino acids 364-550) fused to a CARD domain (NF-κB-AD/CARD). TheGal4-BD/CARD-5 vector, the NF-κB-AD/CARD domain vector library, and aluciferase reporter construct with an upstream Gal4 binding site wereintroduced into human 293T embryonic kidney cells. If a given CARDdomain expressed fused to the NF-κB transcriptional activation domaininteracts with the CARD domain of CARD-5, the NF-κB transcriptionalactivation domain will be brought into proximity with the promotercontrolling luciferase expression, activating luciferase expression andpermitting detection of the interaction. This analysis revealed that theCARD domain of CARD-5 interacts with the CARD domain of caspase-1,CARD-7, and CARD-5 itself.

CARD-7 contains a C-terminal CARD domain as well as an N-terminal pyrindomain, a nucleotide binding site, and a leucine rich repeat region.Detailed information concerning CARD-7 can be found in U.S. applicationSer. No. 09/428,252, filed Oct. 27, 1999, the content of which isincorporated herein by reference.

Caspase-1 promotes inflammation by processing an inactive cytokineprecursor, e.g., the IL-1β precursor, into an active proinflammatorycytokine. Caspase-1 also plays a role in the activation of apoptosis.The caspase-1 polypeptide is initially synthesized in an inactive formthat becomes activated following an oligomerization event mediatedthrough its N-terminal CARD domain. An upstream activator of caspase-1may possess a CARD domain that interacts with the CARD domain ofcaspase-1 thereby leading to its oligomerization. Furthermore, a decoymolecule that attenuates caspase-1 activation may also interact withcaspase-1 via a CARD-CARD interaction.

The finding that the C-terminal CARD domain of CARD-5 binds to the CARDdomain of caspase-1 suggests that CARD-5 may be a modulator of caspase-1activity. For example, CARD-5 may function as an upstream activatoradaptor of caspase-1. Alternatively, CARD-5 binding to caspase-1 mayattenuate the caspase-1 activation mediated by other upstream adaptors.

CARD-5 also possess an N-terminal pyrin domain. An upstream pyrin domaincontaining protein may thus bind to CARD-5, functioning as a regulatorof CARD-5 in the context of a caspase-1 regulation pathway. For example,the upstream pyrin domain containing protein may engage theCARD-5/caspase-1 complex, e.g., via the pyrin domain of CARD-5, andthereby activate caspase-1 via its oligomerization. Examples of pyrindomain containing proteins, and possible upstream regulators of CARD-5,include NBS-1 and Pyrin-1. Detailed information concerning NBS-1 andPyrin-1 can be found in U.S. application Ser. No. 09/506,067, filed Feb.17, 2000, and U.S. application Ser. No. 09/653.901, filed Sep. 1, 2000.The entire content each of these applications is incorporated herein byreference.

Example 18 NF-κB Activation by CARD-5

The binding of CARD-5 to caspase-1 described above suggests thatCARD-5-caspase-1 interactions may be part of a signaling pathwayinvolved in inflammation, apoptosis, and/or NF-κB activation. Consistentwith this signal transduction model, CARD-5 was shown to be an inducerof NF-κB activation. Expression of CARD-5 in 293T cells resulted in a20-30 fold increase in NF-κB activity.

The NF-κB activity assay was performed as described in Example 9. AnNF-κB reporter plasmid was co-transfected with a construct encodingCARD-5. In the reporter plasmid, the luciferase gene was placed underthe control of the NF-κB promoter. Relative luciferase activity wasdetermined at the end of the experiment to assess NF-κB pathwayactivation by CARD-5.

Example 19 Deposit of Clones

A plasmid containing a cDNA encoding human CARD-3 (pXE17A) was depositedwith the American Type Culture Collection (ATCC), Manasass, Va. on May14, 1998. and assigned Accession Number 203037.

A plasmid containing a cDNA encoding human CARD-4L (pC4L1) was depositedwith the American Type Culture Collection (ATCC), Manasass, Va. on Jul.7, 1998, and assigned Accession Number 203035.

A plasmid containing a cDNA encoding human CARD-4S (pDB33E) wasdeposited with the American Type Culture Collection (ATCC), Manasass,Va. on May 14, 1998, and assigned Accession Number 203036.

A plasmid containing a cDNA encoding murine CARD-5 (EpMC5) was depositedwith the American Type Culture Collection (ATCC), Manasass, Va. on Jun.11, 1999, and assigned Accession Number PTA-212.

A plasmid containing a cDNA encoding rat CARD-6 (EpRC6) was depositedwith the American Type Culture Collection (ATCC), Manassas, Va. on Jun.11, 1999, and assigned Accession Number PTA-211.

A clone (EpHC5) containing a cDNA molecule encoding human CARD-5, aclone (EpCH6e) containing a cDNA molecule encoding an amino terminalportion of human CARD-6, a clone (EpHC6c) containing a cDNA moleculeencoding a carboxy terminal portion of human CARD-6, and a clone (EpHC6)containing a cDNA molecule encoding human CARD-6 were deposited with theAmerican Type Culture Collection (ATCC) Manassas, Va. on Jun. 11, 1999,as a composite deposit and assigned Accession Number PTA-213. Todistinguish the strains and isolate a strain harboring a particular cDNAclone, one can first streak out an aliquot of the mixture to singlecolonies on nutrient medium (e.g., LB plates) supplemented with 100μg/ml ampicillin, grow single colonies, and then extract the plasmid DNAfrom a selected colony using a standard minipreparation procedure. Next,one can digest a sample of the DNA minipreparation with a combination ofthe restriction enzymes Sal I and Not I and resolve the resultantproducts on a 0.8% agarose gel using standard DNA electrophoresisconditions. The digestion will liberate DNA fragments as follows: HumanCARD-5 (EpHC5) 0.6 kb and 3.0 kb Human CARD-6 amino- 1.0 kb and 4.3 kbterminal portion (EpHC6e) (amino acids 1-279) Human CARD-6 carboxy 3.8kb and 3.0 kb terminal portion (EpHC6c) (amino acid 93-1037) HumanCARD-6 (EpHC6) 4.2 kb and 3.0 kb (amino acids 1-1037)Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule comprising a nucleotide sequence which isat least 55% identical to the nucleotide sequence of SEQ ID NO:1, 3, 7,9, 25, 27, 38, 40, 42, 48, 50, 51, 53, 54, 56, 60, 62, or the cDNAinsert of the plasmid deposited with the ATCC as any of AccessionNumbers ______, or a complement thereof; b) a nucleic acid moleculecomprising a fragment of at least 300 nucleotides of the nucleotidesequence of SEQ ID NO:1, 3, 7, 9, 25, 27, 38, 40, 42, 48, 50, 51, 53,54, 56, 60, 62, or the cDNA insert of the plasmid deposited with theATCC as any of Accession Numbers ______, or a complement thereof; c) anucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55, 61, oramino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as any of Accession Numbers ______; d) a nucleic acidmolecule which encodes a fragment of a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55, 61, or thepolypeptide encoded by the cDNA insert of the plasmid deposited with theATCC as any of Accession Numbers ______, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:2, 8, 26, 39, 41, 43,49, 52, 55, 61, or the polypeptide encoded by the cDNA insert of theplasmid deposited with the ATCC as any of Accession Numbers ______; ande) a nucleic acid molecule which encodes a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, 8, 26, 39, 41, 43, 49, 52, 55, 61, or the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with the ATCC as anyof Accession Numbers ______, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule comprising SEQ ID NO:1, 3, 7, 9,25, 27, 38, 40, 42, 48, 50, 51, 53, 54, 56, 60, 62, or a complementthereof under stringent conditions.
 2. The isolated nucleic acidmolecule of claim 1, which is selected from the group consisting of: a)a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, 3, 7,9, 25, 27, 38, 40, 42, 48, 50, 51, 53, 54, 56, 60, 62, or the cDNAinsert of the plasmid deposited with the ATCC as any of AccessionNumbers ______, or a complement thereof; and b) a nucleic acid moleculewhich encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:2, 8, 26, 39, 41, 43, 49, 52, 55, 61, or the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with the ATCC as anyof Accession Numbers ______.
 3. The nucleic acid molecule of claim 1further comprising vector nucleic acid sequences.
 4. The nucleic acidmolecule of claim 1 further comprising nucleic acid sequences encoding aheterologous polypeptide.
 5. A host cell which contains the nucleic acidmolecule of claim
 1. 6. The host cell of claim 5 which is a mammalianhost cell.
 7. A non-human mammalian host cell containing the nucleicacid molecule of claim
 1. 8. An isolated polypeptide selected from thegroup consisting of: a) a fragment of a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55, or 61,wherein the fragment comprises at least 15 contiguous amino acids of SEQID NO:2, 8, 26, 39, 41, 43, 49, 52, 55, or 61; b) a naturally occurringallelic variant of a polypeptide comprising the amino acid sequence ofSEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55, or 61, or the amino acidsequence encoded by the cDNA insert of the plasmid deposited with theATCC as any of Accession Numbers ______, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:1, 3, 7, 9, 25, 27, 38, 40, 42, 48, 50,51, 53, 54, 56, 60, 62, or a complement thereof under stringentconditions; and c) a polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence which is at least 65%identical to a nucleic acid comprising the nucleotide sequence of SEQ IDNO:1, 3, 7, 9, 25, 27, 38, 40, 42, 48, 50, 51, 53, 54, 56, 60, 62, or acomplement thereof.
 9. The isolated polypeptide of claim 8 comprisingthe amino acid sequence of SEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55,or
 61. 10. The polypeptide of claim 8 further comprising heterologousamino acid sequences.
 11. An antibody which selectively binds to apolypeptide of claim
 8. 12. A method for producing a polypeptideselected from the group consisting of: a) a polypeptide comprising theamino acid sequence of SEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55, 61,or the amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as any of Accession Numbers ______; b) apolypeptide comprising a fragment of the amino acid sequence of SEQ IDNO:2, 8, 26, 39, 41, 43, 49, 52, 55, 61, or the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with the ATCC as anyof Accession Number ______, wherein the fragment comprises at least 15contiguous amino acids of SEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55,61, or the amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as any of Accession Numbers ______; and c) anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, 8, 26, 39, 41, 43, 49, 52, 55, 61,or the amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as any of Accession Numbers ______, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule comprising SEQ ID NO:1, 3, 7, 9, 25, 27, 38, 40,42, 48, 50, 51, 53, 54, 56, 60, 62, or a complement thereof understringent conditions; comprising culturing the host cell of claim 5under conditions in which the nucleic acid molecule is expressed.
 13. Amethod for detecting the presence of a polypeptide of claim 8 in asample, comprising: a) contacting the sample with a compound whichselectively binds to a polypeptide of claim 8; and b) determiningwhether the compound binds to the polypeptide in the sample.
 14. Themethod of claim 13, wherein the compound which binds to the polypeptideis an antibody.
 15. A kit comprising a compound which selectively bindsto a polypeptide of claim 8 and instructions for use.
 16. A method fordetecting the presence of a nucleic acid molecule of claim 1 in asample, comprising the steps of: a) contacting the sample with a nucleicacid probe or primer which selectively hybridizes to the nucleic acidmolecule; and b) determining whether the nucleic acid probe or primerbinds to a nucleic acid molecule in the sample.
 17. The method of claim16, wherein the sample comprises mRNA molecules and is contacted with anucleic acid probe.
 18. A kit comprising a compound which selectivelyhybridizes to a nucleic acid molecule of claim 1 and instructions foruse.
 19. A method for identifying a compound which binds to apolypeptide of claim 8 comprising the steps of: a) contacting apolypeptide, or a cell expressing a polypeptide of claim 8 with a testcompound; and b) determining whether the polypeptide binds to the testcompound.
 20. The method of claim 19, wherein the binding of the testcompound to the polypeptide is detected by a method selected from thegroup consisting of: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; c) detection of binding using an assay forCARD-3, CARD-4, CARD-5, or CARD-6-mediated signal transduction.
 21. Amethod for modulating the activity of a polypeptide of claim 8comprising contacting a polypeptide or a cell expressing a polypeptideof claim 8 with a compound which binds to the polypeptide in asufficient concentration to modulate the activity of the polypeptide.22. A method for identifying a compound which modulates the activity ofa polypeptide of claim 8, comprising: a) contacting a polypeptide ofclaim 8 with a test compound; and b) determining the effect of the testcompound on the activity of the polypeptide to thereby identify acompound which modulates the activity of the polypeptide.